Nucleic acid molecules and uses thereof

ABSTRACT

The present invention is directed to an artificial nucleic acid and to polypeptides suitable for use in treatment or prophylaxis of an infection with Norovirus or a disorder related to such an infection. In particular, the present invention concerns a Norovirus vaccine. The present invention is directed to an artificial nucleic acid, polypeptides, compositions and vaccines comprising the artificial nucleic acid or the polypeptides. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial nucleic acid, polypeptides, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial nucleic acid, polypeptides, compositions and vaccines.

INTRODUCTION

The present invention is directed to an artificial nucleic acid and topolypeptides suitable for use in treatment or prophylaxis of aninfection with Norovirus or a disorder related to such an infection. Inparticular, the present invention concerns a Norovirus vaccine. Thepresent invention is directed to an artificial nucleic acid,polypeptides, compositions and vaccines comprising the artificialnucleic acid or the polypeptides. The invention further concerns amethod of treating or preventing a disorder or a disease, first andsecond medical uses of the artificial nucleic acid, polypeptides,compositions and vaccines. Further, the invention is directed to a kit,particularly to a kit of parts, comprising the artificial nucleic acid,polypeptides, compositions and vaccines.

Noroviruses (also known as Norwalk-like viruses or Norwalk viruses) arepositive sense, single-stranded RNA Calciviruses (Sarvestani, SoroushT., et al. “Norovirus Infection: Replication, Manipulation of Host, andInteraction with the Host Immune Response.” Journal of Interferon 6Cytokine Research 36.4 (2016):215-225), containing a non-segmented RNAgenome. The virus genome is organized in three open reading frames, ofwhich the 5′ proximal ORF encodes a large polyprotein that is cleavedinto non-structural proteins; the minor capsid protein VP2 is encoded byORF3 and the major capsid protein VP1 is encoded by ORF2 (Karst et al.,Cell Host Microbe 11; 15(6):668-80, 2014; Robilotti E, Deresinski S,Pinsky B A. 2015. Norovirus. Clin Microbiol Rev 28:134-164).

Noroviruses are classified into five genogroups (GI-GV), and are furthersubdivided into genotypes based on the capsid sequence (Zheng, Du-Ping,et al. “Norovirus classification and proposed strain nomenclature.”Virology 346.2 (2001):312-323; Kroneman, A., et al. “An automatedgenotyping tool for enteroviruses and noroviruses.” Journal of ClinicalVirology 51.2 (2011):121-125). Mostly viruses of genogroups I, II areknown to infect humans (Ramani, Sasirekha, Robert L. Atmar, and Mary K.Estes. “Epidemiology of human noroviruses and updates on vaccinedevelopment.”, Current opinion in gastroenterology 30.1 (2014):25).Norwalk viruses (NV) genotype GI.1 was the first isolated Norovirus,however genotype GII.4 Noroviruses are currently the most frequentlydetected in humans (Glass, Roger I., Umesh D. Parashar, and Mary K.Estes. “Norovirus gastroenteritis.” New England Journal of Medicine361.18 (2009):1776-1785).

Infections with Noroviruses are generally self-limiting in healthyadults, displaying typical symptoms including non-bloody diarrhea,vomiting, nausea and abdominal cramps. However, in individuals withweakened immune system, including young children and elderly, infectioncan be severe and even fatal (Glass, Roger I., Umesh D. Parashar, andMary K. Estes. “Norovirus gastroenteritis.” New England Journal ofMedicine 361.18 (2009):1776-1785). Nongastrointestinal-related illness,including neurodevelopmental disorders have also been reported afterNorovirus infection. (Sarvestani, Soroush T., et al. “NorovirusInfection: Replication, Manipulation of Host, and Interaction with theHost Immune Response.” Journal of Interferon C Cytokine Research 36.4(2016):215-225).

Due to the low infectious dose, resistance to many common sterilizationprocedures, and ease of transmission, epidemic outbreaks are common anddifficult to control (Glass, Roger I., Umesh D. Parashar, and Mary K.Estes. “Norovirus gastroenteritis.” New England Journal of Medicine361.18 (200B):1776-1785; Hutson et al., 2004). NoV has been shown to bethe cause of the majority of nonbacterial gastroenteritis epidemics,resulting in a huge economic burden. In the US, the cost ofNoV-associated hospitalizations has been estimated at approximately $500million, while foodborne NoV cost due to healthcare and lostproductivity has been estimated at $2 billion (Batz, Michael B., SandraHoffmann, and J. Glenn Morris Jr. “Ranking the disease burden of 14pathogens in food sources in the United States using attribution datafrom outbreak investigations and expert elicitation.” Journal of FoodProtection 75.7 (2012):1278-1291). The rapid evolution of Norovirusgenotypes through antigenic drift and changing glycan specificitiesstill provide challenges in the development of potent vaccines (Ramani,Sasirekha, Robert L. Atmar, and Mary K. Estes. “Epidemiology of humannoroviruses and updates on vaccine development.” Current opinion ingastroenterology 30.1 (2014):25).

At present, there is no specific treatment of Norovirus infections.Therapy is limited to curing the symptoms caused by the infection. Inaddition, there is currently no vaccine available against Norovirusinfections. There is therefore a strong need for a vaccine againstNorovirus infection.

The underlying object of the present invention is therefore to provide aNorovirus vaccine. It is a further preferred object of the invention toprovide a Norovirus vaccine, which may be produced at an industrialscale. A further object of the present invention is the provision of astorage-stable Norovirus vaccine. Further object of the underlyinginvention is to provide mRNA sequences coding for antigenic peptides orproteins derived from a protein of a Norovirus or a fragment or variantthereof for the use as a vaccine for prophylaxis or treatment ofNorovirus infections. Furthermore, it is the object of the presentinvention to provide an effective Norovirus vaccine which can be storedwithout cold chain and which enables rapid and scalable vaccineproduction.

The object underlying the present invention is solved by the claimedsubject-matter. Particularly, the objects underlying the presentinvention are solved according to a first aspect by an inventive byproviding an artificial nucleic acid comprising at least one codingregion encoding at least one polypeptide derived from a Norovirus,and/or a fragment or variant thereof.

Definitions

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Adaptive immune response: The adaptive immune response is typicallyunderstood to be an antigen-specific response of the immune system.Antigen specificity allows for the generation of responses that aretailored to specific pathogens or pathogen-infected cells. The abilityto mount these tailored responses is usually maintained in the body by“memory cells”. Should a pathogen infect the body more than once, thesespecific memory cells are used to quickly eliminate it. In this context,the first step of an adaptive immune response is the activation of naïveantigen-specific T cells or different immune cells able to induce anantigen-specific immune response by antigen-presenting cells. Thisoccurs in the lymphoid tissues and organs through which naïve T cellsare constantly passing. The three cell types that may serve asantigen-presenting cells are dendritic cells, macrophages, and B cells.Each of these cells has a distinct function in eliciting immuneresponses. Dendritic cells may take up antigens by phagocytosis andmacropinocytosis and may become stimulated by contact with e.g. aforeign antigen to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents or other appropriate stimuli to express MHC molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may also be important to induce T cells. MHC-moleculesare, typically, responsible for presentation of an antigen to T-cells.Therein, presenting the antigen on MHC molecules leads to activation ofT cells which induces their proliferation and differentiation into armedeffector T cells. The most important function of effector T cells is thekilling of infected cells by CD8+ cytotoxic T cells and the activationof macrophages by Th1 cells which together make up cell-mediatedimmunity, and the activation of B cells by both Th2 and Th1 cells toproduce different classes of antibody, thus driving the humoral immuneresponse. T cells recognize an antigen by their T cell receptors whichdo not recognize and bind the antigen directly, but instead recognizeshort peptide fragments e.g. of pathogen-derived protein antigens, e.g.so-called epitopes, which are bound to MHC molecules on the surfaces ofother cells.

Adaptive immune system: The adaptive immune system is essentiallydedicated to eliminate or prevent pathogenic growth. It typicallyregulates the adaptive immune response by providing the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered. The system is highly adaptable becauseof somatic hypermutation (a process of accelerated somatic mutations),and V(D)J recombination (an irreversible genetic recombination ofantigen receptor gene segments). This mechanism allows a small number ofgenes to generate a vast number of different antigen receptors, whichare then uniquely expressed on each individual lymphocyte. Because thegene rearrangement leads to an irreversible change in the DNA of eachcell, all of the progeny (offspring) of such a cell will then inheritgenes encoding the same receptor specificity, including the Memory Bcells and Memory T cells that are the keys to long-lived specificimmunity.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a pharmacological and/or immunological agentthat may modify, e.g. enhance, the effect of other agents, such as adrug or vaccine. It is to be interpreted in a broad sense and refers toa broad spectrum of substances. Typically, these substances are able toincrease the immunogenicity of antigens. For example, adjuvants may berecognized by the innate immune systems and, e.g., may elicit an innateimmune response. “Adjuvants” typically do not elicit an adaptive immuneresponse. Insofar, “adjuvants” do not qualify as antigens. Their mode ofaction is distinct from the effects triggered by antigens resulting inan adaptive immune response.

Antigen: In the context of the present invention “antigen” referstypically to a substance which may be recognized by the immune system,preferably by the adaptive immune system, and is capable of triggeringan antigen-specific immune response, e.g. by formation of antibodiesand/or antigen-specific T cells as part of an adaptive immune response.Typically, an antigen may be or may comprise a peptide or protein whichmay be presented by the MHC to T-cells. In the sense of the presentinvention an antigen may be the product of translation of a providednucleic acid molecule, preferably an mRNA as defined herein. In thiscontext, also fragments, variants and derivatives of peptides andproteins comprising at least one epitope are understood as antigens. Inthe context of the present invention, tumour antigens and pathogenicantigens as defined herein are particularly preferred.

Artificial nucleic acid molecule: An “artificial nucleic acid molecule”or “artificial nucleic acid” may typically be understood to be a nucleicacid molecule, e.g. a DNA or an RNA that does not occur naturally. Inother words, an artificial nucleic acid molecule may be understood as anon-natural nucleic acid molecule. Such nucleic acid molecule may benon-natural due to its individual sequence (which does not occurnaturally) and/or due to other modifications, e.g. structuralmodifications of nucleotides which do not occur naturally. An artificialnucleic acid molecule may be a DNA molecule, an RNA molecule or ahybrid-molecule comprising DNA and RNA portions. Typically, artificialnucleic acid molecules may be designed and/or generated by geneticengineering methods to correspond to a desired artificial sequence ofnucleotides (heterologous sequence). In this context an artificialsequence is usually a sequence that may not occur naturally, i.e. itdiffers from the wild type sequence by at least one nucleotide. The term“wild type” may be understood as a sequence occurring in nature.Further, the term “artificial nucleic acid molecule” is not restrictedto mean “one single molecule” but is, typically, understood to comprisean ensemble of identical molecules. Accordingly, it may relate to aplurality of identical molecules contained in an aliquot.

Bicistronic nucleic acid or RNA and multicistronic nucleic acid or RNA:A bicistronic or multicistronic nucleic acid or RNA is typically anucleic acid or an RNA, preferably an mRNA, that typically may have two(bicistronic) or more (multicistronic) coding regions. A coding regionin this context is a sequence of codons that is translatable into apeptide or protein.

Carrier/polymeric carrier: A carrier in the context of the invention maytypically be a compound that facilitates transport and/or complexationof another compound (cargo). A polymeric carrier is typically a carrierthat is formed of a polymer. A carrier may be associated to its cargo bycovalent or non-covalent interaction. A carrier may transport nucleicacids, e.g. RNA or DNA, to the target cells. The carrier may—for someembodiments—be a cationic component.

Complexation and Formulation: According to a preferred embodiment, theat least one mRNA of the inventive composition may be complexed withlipids to form one or more liposomes, lipoplexes, or lipidnanoparticles. Therefore, in one embodiment, the inventive compositioncomprises liposomes, lipoplexes, and/or lipid nanoparticles comprisingthe at least one mRNA.

Lipid-based formulations have been increasingly recognized as one of themost promising delivery systems for RNA due to their biocompatibilityand their ease of large-scale production. Cationic lipids have beenwidely studied as synthetic materials for delivery of RNA. After mixingtogether, nucleic acids are condensed by cationic lipids to formlipid/nucleic acid complexes known as lipoplexes. These lipid complexesare able to protect genetic material from the action of nucleases anddeliver it into cells by interacting with the negatively charged cellmembrane. Lipoplexes can be prepared by directly mixing positivelycharged lipids at physiological pH with negatively charged nucleicacids. Conventional liposomes consist of a lipid bilayer that can becomposed of cationic, anionic, or neutral (phospho)lipids andcholesterol, which encloses an aqueous core. Both the lipid bilayer andthe aqueous space can incorporate hydrophobic or hydrophilic compounds,respectively. Liposome characteristics and behaviour in vivo can bemodified by addition of a hydrophilic polymer coating, e.g. polyethyleneglycol (PEG), to the liposome surface to confer steric stabilization.Furthermore, liposomes can be used for specific targeting by attachingligands (e.g., antibodies, peptides, and carbohydrates) to its surfaceor to the terminal end of the attached PEG chains (Front Pharmacol. 2015Dec. 1:6:286). Liposomes are colloidal lipid-based and surfactant-baseddelivery systems composed of a phospholipid bilayer surrounding anaqueous compartment. They may present as spherical vesicles and canrange in size from 20 nm to a few microns. Cationic lipid-basedliposomes are able to complex with negatively charged nucleic acids viaelectrostatic interactions, resulting in complexes that offerbiocompatibility, low toxicity, and the possibility of the large-scaleproduction required for in vivo clinical applications. Liposomes canfuse with the plasma membrane for uptake: once inside the cell, theliposomes are processed via the endocytic pathway and the geneticmaterial is then released from the endosome/carrier into the cytoplasm.Liposomes have long been perceived as drug delivery vehicles because oftheir superior biocompatibility, given that liposomes are basicallyanalogs of biological membranes, and can be prepared from both naturaland synthetic phospholipids (Int J Nanomedicine. 2014; 9:1833-1843).

Cationic liposomes have been traditionally the most commonly usednon-viral delivery systems for oligonucleotides, including plasmid DNA,antisense oligos, and siRNA/small hairpin RNA-shRNA). Cationic lipids,such as DOTAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate)can form complexes or lipoplexes with negatively charged nucleic acidsto form nanoparticles by electrostatic interaction, providing high invitro transfection efficiency. Furthermore, neutral lipid-basednanoliposomes for RNA delivery as e.g. neutral1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomeswere developed. (Adv Drug Deliv Rev. 2014 February: 66:110-116).

Therefore, in one embodiment the at least one mRNA of the inventivecomposition is complexed with cationic lipids and/or neutral lipids andthereby forms liposomes, lipid nanoparticles, lipoplexes or neutrallipid-based nanoliposomes.

Cationic component or cationic compound: The term “cationic component”or “cationic compound” typically refers to a charged molecule, which ispositively charged (cation) at a pH value typically from 1 to 9,preferably at a pH value of or below 9 (e.g. from 5 to 9), of or below 8(e.g. from 5 to 8), of or below 7 (e.g. from 5 to 7), most preferably ata physiological pH, e.g. from 7.3 to 7.4. Accordingly, a cationiccomponent may be any positively charged compound or polymer, preferablya cationic peptide or protein which is positively charged underphysiological conditions, particularly under physiological conditions invivo. Further accordingly, a cationic peptide, protein, polysaccharide,lipid or polymer according to the present invention is positivelycharged under physiological conditions, particularly under physiologicalsalt conditions of the cell in vivo. A “cationic peptide or protein” maycontain at least one positively charged amino acid, or more than onepositively charged amino acid, e.g. selected from Arg, His, Lys or Orn.Accordingly, “polycationic” components or compounds are also within thescope exhibiting more than one positive charge under the conditionsgiven.

5′-cap: A 5′-cap is an entity, typically a modified nucleotide entity,which generally “caps” the 5′-end of a mature mRNA. A 5′-cap maytypically be formed by a modified nucleotide, particularly by aderivative of a guanine nucleotide. Preferably, the 5′-cap is linked tothe 5′-terminus via a 5′-5′-triphosphate linkage. A 5′-cap may bemethylated, e.g. m7GpppN, wherein N is the terminal 5′ nucleotide of thenucleic acid carrying the 5′-cap, typically the 5′-end of an RNA.Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1.5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide. 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety.

Cap analogue: A cap analogue refers to a non-polymerizable di-nucleotidethat has cap functionality in that it facilitates translation orlocalization, and/or prevents degradation of a nucleic acid molecule,particularly of an RNA molecule, when incorporated at the 5′ end of thenucleic acid molecule. Non-polymerizable means that the cap analoguewill be incorporated only at the 5′-terminus because it does not have a5′ triphosphate and therefore cannot be extended in the 3′ direction bya template-dependent polymerase, particularly, by template-dependent RNApolymerase.

Cap analogues include, but are not limited to, a chemical structureselected from the group consisting of m7GpppG, m7GpppA, m7GpppC;unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue(e.g., m2.7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG),dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or antireverse cap analogues (e.g., ARCA: m7.2′OmeGpppG. m7.2′dGpppG,m7.3′OmeGpppG, m7.3′dGpppB and their tetraphosphate derivatives)(Stepinski et al., 2001. RNA 7(10):1486-95).

Further cap analogues have been described previously (U.S. Pat. No.7,074,596, WO 2008/016473, WO 2008/157688, WO 2009/149253. WO2011/015347, and WO 2013/059475). The synthesis ofN7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogues hasbeen described recently (Kore et al. (2013) Bioorg. Med. Chem.21(15):4571-4).

5′-cap-Structure: A 5′-cap is typicallyα a modified nucleotide (capanalogue), particularly a guanine nucleotide, added to the 5′ end of anucleic acid molecule, particularly of an RNA molecule, e.g. an mRNAmolecule. Preferably, the 5′-cap is added using a 5′-5′-triphosphatelinkage (also named m7GpppN). Further examples of 5′-cap structuresinclude glyceryl, inverted deoxy abasic residue (moiety), 4′,5′methylene nucleotide. 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,L-nucleotides, alpha-nucleotide, modified base nucleotide,threo-pentofuranosyl nucleotide, acyclic 3′,4′-seco nucleotide, acyclic3,4-dihydroxybutyl nucleotide, acyclic 3.5 dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety,3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety,1,4-butanedial phosphate, 3′-phosphoramidate, hexylphosphate, aminohexylphosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, orbridging or non-bridging methylphosphonate moiety. These modified 5′-capstructures may be used in the context of the present invention to modifythe mRNA sequence of the inventive composition. Further modified 5′-capstructures which may be used in the context of the present invention arecap 1 (additional methylation of the ribose of the adjacent nucleotideof m77GpppN), cap2 (additional methylation of the ribose of the 2ndnucleotide downstream of the m7GpppN), cap3 (additional methylation ofthe ribose of the 3rd nucleotide downstream of the m7GpppN), cap4(additional methylation of the ribose of the 4th nucleotide downstreamof the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

In the context of the present invention, a 5′ cap structure (cap0 orcap1) may also be formed in chemical RNA synthesis or RNA in vitrotranscription (co-transcriptional capping) using cap analogues, or a capstructure may be formed in vitro using capping enzymes (e.g.,commercially available capping kits). A cap structure (e.g., cap0 orcap1) may also be formed in vitro using immobilized capping enzymes,e.g. in a capping reactor as described in WO 2016/19322.

Chemical synthesis of nucleic acids: Chemical synthesis of relativelyshort fragments of oligonucleotides with defined chemical structureprovides a rapid and inexpensive access to custom-made oligonucleotidesof any desired sequence. Whereas enzymes synthesize DNA and RNA only inthe 5′ to 3′ direction, chemical oligonucleotide synthesis does not havethis limitation, although it is most often carried out in the opposite,i.e. the 3′ to 5′ direction. Currently, the process is implemented assolid-phase synthesis using the phosphoramidite method andphosphoramidite building blocks derived from protected nucleosides (A,C, G, and U), or chemically modified nucleosides.

To obtain the desired oligonucleotide, the building blocks aresequentially coupled to the growing oligonucleotide chain on a solidphase in the order required by the sequence of the product in a fullyautomated process. Upon the completion of the chain assembly, theproduct is released from the solid phase to the solution, deprotected,and collected. The occurrence of side reactions sets practical limitsfor the length of synthetic oligonucleotides (up to about 200 nucleotideresidues), because the number of errors increases with the length of theoligonucleotide being synthesized. Products are often isolated by HPLCto obtain the desired oligonucleotides in high purity.

Chemically synthesized oligonucleotides find a variety of applicationsin molecular biology and medicine. They are most commonly used asantisense oligonucleotides, small interfering RNA, primers for DNAsequencing and amplification, probes for detecting complementary DNA orRNA via molecular hybridization, tools for the targeted introduction ofmutations and restriction sites, and for the synthesis of artificialgenes. Moreover, long-chain DNA molecules and long-chain RNA moleculesmay be chemically synthetized and used in the context of the presentinvention.

Cellular immunity/cellular immune response: Cellular immunity relatestypically to the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes, and the release of variouscytokines in response to an antigen. In more general terms, cellularimmunity is not based on antibodies, but on the activation of cells ofthe immune system. Typically, a cellular immune response may becharacterized e.g. by activating antigen-specific cytotoxicT-lymphocytes that are able to induce apoptosis in cells, e.g. specificimmune cells like dendritic cells or other cells, displaying epitopes offoreign antigens on their surface. Such cells may be virus-infected orinfected with intracellular bacteria, or cancer cells displaying tumorantigens. Further characteristics may be activation of macrophages andnatural killer cells, enabling them to destroy pathogens and stimulationof cells to secrete a variety of cytokines that influence the functionof other cells involved in adaptive immune responses and innate immuneresponses.

Cloning site: A cloning site is typically understood to be a segment ofa nucleic acid molecule, which is suitable for insertion of a nucleicacid sequence, e.g., a nucleic acid sequence comprising a coding region.Insertion may be performed by any molecular biological method known tothe one skilled in the art, e.g. by restriction and ligation. A cloningsite typically comprises one or more restriction enzyme recognitionsites (restriction sites). These one or more restrictions sites may berecognized by restriction enzymes which cleave the DNA at these sites. Acloning site which comprises more than one restriction site may also betermed a multiple cloning site (MCS) or a polylinker.

Coding region, coding sequence: A coding region, in the context of theinvention, is typically a sequence of several nucleotide triplets, whichmay be translated into a peptide or protein. A coding region preferablycontains a start codon, i.e. a combination of three subsequentnucleotides coding usually for the amino acid methionine (ATG), at its5′-end and a subsequent region which usually exhibits a length which isa multiple of 3 nucleotides. A coding region is preferably terminated bya stop-codon (e.g., TAA, TAG, TGA). Typically, this is the onlystop-codon of the coding region. Thus, a coding region in the context ofthe present invention is preferably a nucleotide sequence, consisting ofa number of nucleotides that may be divided by three, which starts witha start codon (e.g. ATG) and which preferably terminates with a stopcodon (e.g., TAA, TGA, or TAG). The coding region may be isolated or itmay be incorporated in a longer nucleic acid sequence, for example in avector or an mRNA. In the context of the present invention, a codingregion may also be termed “protein coding region”, “coding sequence”,“CDS”, “open reading frame” or “ORF”.

Derived from: The phrase “derived from” as used throughout the presentspecification in the context of a nucleic acid, i.e. for a nucleic acid“derived from” (another) nucleic acid, means that the nucleic acid,which is derived from (another) nucleic acid, shares at least 50%,preferably at least 60%, preferably at least 70%, more preferably atleast 75%, more preferably at least 80%, more preferably at least 85%,even more preferably at least 90%, even more preferably at least 95%,and particularly preferably at least 98% sequence identity with thenucleic acid from which it is derived. In one embodiment, “derived from”means having in increasing order of preference at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 80%, 61%, 62%, 63%, 84%, 65%, 66%,87%, 88%, 89%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 968%, 97%, 98%, or 99% or more sequence identity to the sequencesas represented by SEQ ID NOs: 1-39690, 39713-39748. The skilled personis aware that sequence identity is typically calculated for the sametypes of nucleic acids, i.e. for DNA sequences or for RNA sequences.Thus, it is understood, if a DNA is “derived from” an RNA or if an RNAis “derived from” a DNA, in a first step the RNA sequence is convertedinto the corresponding DNA sequence (in particular by replacing theuracils (U) by thymidines (T) throughout the sequence) or, vice versa,the DNA sequence is converted into the corresponding RNA sequence (inparticular by replacing the thymidines (T) by uracils (U) throughout thesequence). Thereafter, the sequence identity of the DNA sequences or thesequence identity of the RNA sequences is determined. Preferably, anucleic acid “derived from” a nucleic acid also refers to nucleic acid,which is modified in comparison to the nucleic acid from which it isderived, e.g. in order to increase RNA stability even further and/or toprolong and/or increase protein production. It goes without saying thatsuch modifications are preferred, which do not impair RNA stability,e.g. in comparison to the nucleic acid from which it is derived.

Different Noro virus: The term “different Noro virus” in the context ofthe invention has to be understood as the difference between at leasttwo respective Noroviruses, wherein the difference is manifested on theRNA genome of the respective different virus. In the broadest sense,“different Norovirus” has to be understood as genetically “differentNorovirus”. Particularly, said (genetically) different Norovirusesexpress at least one different protein or peptide, wherein the at leastone different protein or peptide preferably differs in at least oneamino acid.

Same Norovirus: In the broadest sense, “same Norovirus” has to beunderstood as genetically the same. Particularly, said (genetically)same virus expresses the same proteins or peptides (e.g., at least onestructural and/or non-structural protein), wherein all proteins orpeptides have the same amino acid sequence.

DNA: DNA is the usual abbreviation for deoxy-ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually deoxy-adenosine-monophosphate,deoxy-thymidine-monophosphate. deoxy-guanosine-monophosphate anddeoxy-cytidine-monophosphate monomers which are—by themselves—composedof a sugar moiety (deoxyribose), a base moiety and a phosphate moiety,and polymerize by a characteristic backbone structure. The backbonestructure is, typically, formed by phosphodiester bonds between thesugar moiety of the nucleotide, i.e. deoxyribose, of a first and aphosphate moiety of a second, adjacent monomer. The specific order ofthe monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the DNA sequence. DNA may be singlestranded or double stranded. In the double stranded form, thenucleotides of the first strand typically hybridize with the nucleotidesof the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.

Mono-, bi- and multicistronic and multi-antigen nucleic acids: Amonocistronic nucleic acid may typically be a DNA or RNA, particularlyan mRNA that comprises only one coding sequences. A coding sequence inthis context is a sequence of several nucleotide triplets (codons) thatcan be translated into a peptide or protein

According to a further embodiment the coding region of the at least onemRNA of the composition may encode at least two, three, four, five, six,seven, eight and more antigens (or fragments and derivatives thereof)linked with or without an amino acid linker sequence, wherein saidlinker sequence can comprise rigid linkers, flexible linkers, cleavablelinkers (e.g., self-cleaving peptides) or a combination thereof.Therein, the antigens may be identical or different or a combinationthereof. According to the present invention, specific antigen and/orepitope combinations according to the paragraph “specific antigencombinations” disclosed above are particularly envisioned. Particularantigen/epitope combinations can be encoded by said mRNA encoding atleast two antigens as explained above (herein referred to as“multi-antigen-constructs/mRNA”).

According to a further embodiment the composition of the presentinvention, may comprise a mixture of at least one monocistronic mRNA, asdefined above, and/or at least one bicistronic mRNA as defined above,and/or at least one multicistronic mRNA, as defined above, and/or atleast one multi-antigen-constructs as defined above, and anycombinations thereof. According to the present invention, specificantigen combinations according to the paragraph “specific antigencombinations” disclosed above are particularly envisioned and may begenerated using a combination of mono-, bi-, multicistronic mRNA andmulti-antigen-constructs.

According to certain embodiments of the present invention, the mRNAsequence is mono-, bi-, or multicistronic, preferably as defined herein.The coding sequences in a bi- or multicistronic mRNA preferably encodedistinct peptides or proteins as defined herein or a fragment or variantthereof. Preferably, the coding sequences encoding two or more peptidesor proteins may be separated in the bi- or multicistronic mRNA by atleast one IRES (internal ribosomal entry site) sequence, as definedbelow. Thus, the term “encoding two or more peptides or proteins” maymean, without being limited thereto, that the bi- or even multicistronicmRNA, may encode e.g. at least two, three, four, five, six or more(preferably different) peptides or proteins or their fragments orvariants within the definitions provided herein. More preferably,without being limited thereto, the bi- or even multicistronic mRNA, mayencode, for example, at least two, three, four, five, six or more(preferably different) peptides or proteins as defined herein or theirfragments or variants as defined herein. In this context, a so-calledIRES (internal ribosomal entry site) sequence as defined above canfunction as a sole ribosome binding site, but it can also serve toprovide a bi- or even multicistronic mRNA as defined above, whichencodes several peptides or proteins which are to be translated by theribosomes independently of one another. Examples of IRES sequences,which can be used according to the invention, are those frompicornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV),encephalomyocarditis viruses (ECMV), foot and mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) orcricket paralysis viruses (CrPV).

According to a further embodiment the at least one coding region of themRNA sequence according to the invention may encode at least two, three,four, five, six, seven, eight and more peptides or proteins (orfragments and derivatives thereof) as defined herein linked with orwithout an amino acid linker sequence, wherein said linker sequence cancomprise rigid linkers, flexible linkers, cleavable linkers (e.g.,self-cleaving peptides) or a combination thereof. Therein, the peptidesor proteins may be identical or different or a combination thereof.Particular peptide or protein combinations can be encoded by said mRNAencoding at least two peptides or proteins as explained herein (alsoreferred to herein as “multi-antigen-constructs/mRNA”).

Epitope: (also called “antigen determinant”) can be distinguished in Tcell epitopes and B cell epitopes. T cell epitopes or parts of theproteins in the context of the present invention may comprise fragmentspreferably having a length of about B to about 20 or even more aminoacids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragmentsas processed and presented by MHC class II molecules, preferably havinga length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18,19, 20 or even more amino acids, wherein these fragments may be selectedfrom any part of the amino acid sequence. These fragments are typicallyrecognized by T cells in form of a complex consisting of the peptidefragment and an MHC molecule, i.e. the fragments are typically notrecognized in their native form. B cell epitopes are typically fragmentslocated on the outer surface of (native) protein or peptide antigens asdefined herein, preferably having 5 to 15 amino acids, more preferablyhaving 5 to 12 amino acids, even more preferably having 6 to 9 aminoacids, which may be recognized by antibodies, i.e. in their native form.Such epitopes of proteins or peptides may furthermore be selected fromany of the herein mentioned variants of such proteins or peptides. Inthis context antigenic determinants can be conformational ordiscontinuous epitopes which are composed of segments of the proteins orpeptides as defined herein that are discontinuous in the amino acidsequence of the proteins or peptides as defined herein but are broughttogether in the three-dimensional structure or continuous or linearepitopes which are composed of a single polypeptide chain.

Fragment of a sequence: A fragment of a sequence may typically be ashorter portion of a full-length sequence of e.g. a nucleic acidmolecule or an amino acid sequence. Accordingly, a fragment, typically,consists of a sequence that is identical to the corresponding stretchwithin the full-length sequence. A preferred fragment of a sequence inthe context of the present invention, consists of a continuous stretchof entities, such as nucleotides or amino acids corresponding to acontinuous stretch of entities in the molecule the fragment is derivedfrom, which represents at least 5%, 10%, 20%, preferably at least 301%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 31%, even more preferably at least 70%, and mostpreferably at least 801% of the total (i.e. full-length) molecule fromwhich the fragment is derived.

G/C modified: A G/C-modified nucleic acid may typically be a nucleicacid, preferably an artificial nucleic acid molecule as defined herein,based on a modified wild type sequence comprising a preferably increasednumber of guanosine and/or cytosine nucleotides as compared to the wildtype sequence. Such an increased number may be generated by substitutionof codons containing adenosine or thymidine nucleotides by codonscontaining guanosine or cytosine nucleotides. If the enriched G/Ccontent occurs in a coding region of DNA or RNA, it makes use of thedegeneracy of the genetic code. Accordingly, the codon substitutionspreferably do not alter the encoded amino acid residues, but exclusivelyincrease the G/C content of the nucleic acid molecule.

Gene therapy: Gene therapy may typically be understood to mean atreatment of a patient's body or isolated elements of a patient's body,for example isolated tissues/cells, by nucleic acids encoding a peptideor protein. It typically may comprise at least one of the steps of a)administration of a nucleic acid, preferably an artificial nucleic acidmolecule as defined herein, directly to the patient—by whateveradministration route—or in vitro to isolated cells/tissues of thepatient, which results in transfection of the patient's cells either invivo/ex vivo or in vitro; b) transcription and/or translation of theintroduced nucleic acid molecule: and optionally c) re-administration ofisolated, transfected cells to the patient, if the nucleic acid has notbeen administered directly to the patient.

Genetic vaccination: Genetic vaccination may typically be understood tobe vaccination by administration of a nucleic acid molecule encoding anantigen or an immunogen or fragments thereof. The nucleic acid moleculemay be administered to a subject's body or to isolated cells of asubject. Upon transfection of certain cells of the body or upontransfection of the isolated cells, the antigen or immunogen may beexpressed by those cells and subsequently presented to the immunesystem, eliciting an adaptive, i.e. antigen-specific immune response.Accordingly, genetic vaccination typically comprises at least one of thesteps of a) administration of a nucleic acid, preferably an artificialnucleic acid molecule as defined herein, to a subject, preferably apatient, or to isolated cells of a subject, preferably a patient, whichusually results in transfection of the subject's cells either in vivo orin vitro; b) transcription and/or translation of the introduced nucleicacid molecule; and optionally c) re-administration of isolated,transfected cells to the subject, preferably the patient, if the nucleicacid has not been administered directly to the patient.

Genotype, genotype of a virus: The terms “genotype” or “genotype of avirus” have to be understood as the genetic constitution of anindividual or a group or class of organisms having the same geneticallyconsistent structure. Genotyping means determining differences in thegenetic of an individual. In the context of the invention, Norovirusgenotype has to be understood as a Noro virus having the samegenetically consistent structure.

Heterologous sequence: Two sequences are typically understood to be“heterologous” if they are not derivable from the same gene or in thesame allele. I.e., although heterologous sequences may be derivable fromthe same organism, they naturally (in nature) do not occur in the samenucleic acid molecule, such as in the same mRNA.

Homolog of a nucleic acid sequence: The term “homolog” of a nucleic acidsequence refers to sequences of other species than the particularsequence. It is particularly preferred that the nucleic acid sequence isof human origin and therefore it is preferred that the homolog is ahomolog of a human nucleic acid sequence.

Humoral immunity/humoral immune response: Humoral immunity referstypically to antibody production and optionally to accessory processesaccompanying antibody production. A humoral immune response may betypically characterized, e.g., by Th2 activation and cytokineproduction, germinal center formation and isotype switching, affinitymaturation and memory cell generation. Humoral immunity also typicallymay refer to the effector functions of antibodies, which includepathogen and toxin neutralization, classical complement activation, andopsonin promotion of phagocytosis and pathogen elimination.

Immunogen: In the context of the present invention an immunogen may betypically understood to be a compound that is able to stimulate animmune response. Preferably, an immunogen is a peptide, polypeptide, orprotein. In a particularly preferred embodiment, an immunogen in thesense of the present invention is the product of translation of aprovided nucleic acid molecule, preferably an artificial nucleic acidmolecule as defined herein. Typically, an immunogen elicits at least anadaptive immune response.

Immunostimulatory composition: In the context of the invention, animmunostimulatory composition may be typically understood to be acomposition containing at least one component which is able to induce animmune response or from which a component which is able to induce animmune response is derivable. Such immune response may be preferably aninnate immune response or a combination of an adaptive and an innateimmune response. Preferably, an immunostimulatory composition in thecontext of the invention contains at least one artificial nucleic acidmolecule, more preferably an RNA, for example an mRNA molecule. Theimmunostimulatory component, such as the mRNA may be complexed with asuitable carrier. Thus, the immunostimulatory composition may comprisean mRNA/carrier-complex. Furthermore, the immunostimulatory compositionmay comprise an adjuvant and/or a suitable vehicle for theimmunostimulatory component, such as the mRNA.

Immune response: An immune response may typically be a specific reactionof the adaptive immune system to a particular antigen (so calledspecific or adaptive immune response) or an unspecific reaction of theinnate immune system (so called unspecific or innate immune response),or a combination thereof.

Immune system: The immune system may protect organisms from infection.If a pathogen succeeds in passing a physical barrier of an organism andenters this organism, the innate immune system provides an immediate,but non-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts typically contains socalled humoral and cellular components.

Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the contextof the invention may typically be an RNA that is able to induce aninnate immune response. It usually does not have a coding region andthus does not provide a peptide-antigen or immunogen but elicits animmune response e.g. by binding to a specific kind of Toll-like-receptor(TLR) or other suitable receptors. However, of course also mRNAs havinga coding region and coding for a peptide/protein may induce an innateimmune response and, thus, may be immunostimulatory RNAs.

Innate immune system: The innate immune system, also known asnon-specific (or unspecific) immune system, typically comprises thecells and mechanisms that defend the host from infection by otherorganisms in a non-specific manner. This means that the cells of theinnate system may recognize and respond to pathogens in a generic way,but unlike the adaptive immune system, it does not confer long-lastingor protective immunity to the host. The innate immune system may be,e.g., activated by ligands of Toll-like receptors (TLRs) or otherauxiliary substances such as lipopolysaccharides. TNF-alpha, CD40ligand, or cytokines, monokines, lymphokines, interleukins orchemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-ID,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33. IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of humanToll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand ofa NOD-like receptor, a ligand of a RIG-I like receptor, animmunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), aCpG-DNA, an antibacterial agent, or an anti-viral agent. Thepharmaceutical composition according to the present invention maycomprise one or more such substances. Typically, a response of theinnate immune system includes recruiting immune cells to sites ofinfection, through the production of chemical factors, includingspecialized chemical mediators, called cytokines; activation of thecomplement cascade; identification and removal of foreign substancespresent in organs, tissues, the blood and lymph, by specialized whiteblood cells; activation of the adaptive immune system; and/or acting asa physical and chemical barrier to infectious agents.

Jet injection: The term “jet injection”, as used herein, refers to aneedle-free injection method, wherein a fluid containing at least oneinventive nucleic acid sequence (e.g., RNA, DNA, mRNA) and, optionally,further suitable excipients is forced through an orifice, thusgenerating an ultra-fine liquid stream of high pressure that is capableof penetrating mammalian skin and, depending on the injection settings,subcutaneous tissue or muscle tissue. In principle, the liquid streamforms a hole in the skin, through which the liquid stream is pushed intothe target tissue. Preferably, jet injection is used for intradermal,subcutaneous or intramuscular injection of the mRNA sequence accordingto the invention. In a preferred embodiment, jet injection is used forintramuscular injection of the mRNA sequence according to the invention.In a further preferred embodiment, jet injection is used for intradermalinjection of the mRNA sequence according to the invention.

Monovalent/monovalent vaccine: A monovalent vaccine, also calledunivalent vaccine, is designed against a single antigen for a singleorganism. The term “monovalent vaccine” includes the immunizationagainst a single valence. In the context of the invention, a monovalentNorovirus vaccine would comprise a vaccine comprising an artificialnucleic acid encoding one single antigenic peptide or protein derivedfrom one specific Norovirus strain.

Nucleic acid molecule: A nucleic acid molecule, an artificial nucleicacid, or nucleic acid is a molecule comprising, preferably consisting ofnucleic acid components. The terms nucleic acid molecule, artificialnucleic acid, or nucleic acid preferably refer to DNA or RNA moleculesand vice versa. It is preferably used synonymous with the term“polynucleotide”. Preferably, a nucleic acid molecule is a polymercomprising or consisting of nucleotide monomers, which are covalentlylinked to each other by phosphodiester-bonds of asugar/phosphate-backbone. The terms “nucleic acid molecule”, “artificialnucleic acid” or “nucleic acid” also encompasses modified nucleic acidmolecules, such as base-modified, sugar-modified or backbone-modifiedetc. It encompasses any type of DNA or RNA molecules.

Nucleic acid sequence/amino acid: The sequence of a nucleic acidmolecule is typically understood to be the particular and individualorder, i.e. the succession of its nucleotides. The sequence of a proteinor peptide is typically understood to be the order, i.e. the successionof its amino acids.

Peptide: A peptide or polypeptide is typically a polymer of amino acidmonomers, linked by peptide bonds. It typically contains less than 50monomer units. Nevertheless, the term peptide is not a disclaimer formolecules having more than 50 monomer units. Long peptides are alsocalled polypeptides, typically having between 50 and 600 monomericunits. The term “polypeptide” as used herein, however, is typically notlimited by the length of the molecule it refers to. In the context ofthe present invention, the term “polypeptide” may also be used withrespect to peptides comprising less than 50 (e.g. 10) amino acids orpeptides comprising even more than 600 amino acids. Also, the terms“polypeptide”, “peptide”, and “protein” are used interchangeably hereinto refer to polymers of amino acids of any length.

Pharmaceutically effective amount: A pharmaceutically effective amountin the context of the invention is typically understood to be an amountthat is sufficient to induce a pharmaceutical effect, such as an immuneresponse, altering a pathological level of an expressed peptide orprotein, or substituting a lacking gene product, e.g., in case of apathological situation.

Protein: A protein typically comprises one or more peptides orpolypeptides. A protein is typically folded into 3-dimensional form,which may be required for the protein to exert its biological function.

Poly(A) sequence: A poly(A) sequence, also called poly(A) tail or3′-poly(A) tail, is typically understood to be a sequence of adenosinenucleotides, e.g., of up to about 400 adenosine nucleotides, e.g. fromabout 20 to about 400, preferably from about 50 to about 400, morepreferably from about 50 to about 300, even more preferably from about50 to about 250, most preferably from about 60 to about 250 adenosinenucleotides. A poly(A) sequence is typically located at the 3′-end of anmRNA. In the context of the present invention, a poly(A) sequence may belocated within an mRNA or any other nucleic acid molecule, such as,e.g., in a vector, for example, in a vector serving as template for thegeneration of an RNA, preferably an mRNA, e.g., by transcription of thevector. Moreover, poly(A) sequences, or poly(A) tails may be generatedin vitro by enzymatic polyadenylation of the RNA, e.g. usingPoly(A)polymerases (PAP) derived from E. coli or yeast. In addition,polyadenylation of RNA can be achieved by using immobilized PAP enzymese.g. in a polyadenylation reactor (WO 2016/174271).

Poly(C) sequence: A poly(C) sequence is typically a long sequence ofcytosine nucleotides, typically about 10 to about 21100 cytosinenucleotides, preferably about 10 to about 100 cytosine nucleotides, morepreferably about 10 to about 70 cytosine nucleotides or even more,preferably about 20 to about 50, or even about 20 to about 30 cytosinenucleotides. A poly(C) sequence may preferably be located 3′ of thecoding sequence comprised by a nucleic acid.

Polyadenylation: Polyadenylation is typically understood to be theaddition of a poly(A) sequence to a nucleic acid molecule, such as anRNA molecule, e.g. to a premature mRNA. Polyadenylation may be inducedby a so called polyadenylation signal. This signal is preferably locatedwithin a stretch of nucleotides at the 3′-end of a nucleic acidmolecule, such as an RNA molecule, to be polyadenylated. Apolyadenylation signal typically comprises a hexamer consisting ofadenine and uracil/thymine nucleotides, preferably the hexamer sequenceAAUAAA. Other sequences, preferably hexamer sequences, are alsoconceivable. Polyadenylation typically occurs during processing of apre-mRNA (also called premature-mRNA). Typically, RNA maturation (frompre-mRNA to mature mRNA) comprises the step of polyadenylation.

Polyvalent/polyvalent vaccine: A polyvalent vaccine, called alsomultivalent vaccine, containing antigens from more than one strain of avirus, or different antigens of the same virus, or any combinationthereof. The term “polyvalent vaccine” describes that this vaccine hasmore than one valence. In the context of the invention, a polyvalentNorovirus vaccine would comprise a vaccine comprising an artificialnucleic acid encoding antigenic peptides or proteins derived fromseveral different Norovirus strains or comprising artificial nucleicacid encoding different antigens from the same Norovirus strain, or acombination thereof. In preferred embodiment, a polyvalent Norovirusvaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 88,90, 91, 92, 93, 84, 95, 98, 97, 98, 99, 100 or even more differentartificial nucleic acids each encoding at least one different antigenicpeptide or protein. Methods to produce polyvalent mRNA vaccines aredisclosed in the PCT application PCT/EP2016/82487.

Restriction site: A restriction site, also termed restriction enzymerecognition site, is a nucleotide sequence recognized by a restrictionenzyme. A restriction site is typically a short, preferably palindromicnucleotide sequence, e.g. a sequence comprising 4 to 8 nucleotides. Arestriction site is preferably specifically recognized by a restrictionenzyme. The restriction enzyme typically cleaves a nucleotide sequencecomprising a restriction site at this site. In a double-strandednucleotide sequence, such as a double-stranded DNA sequence, therestriction enzyme typically cuts both strands of the nucleotidesequence.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic-acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually adenosine-monophosphate, uridine-monophosphate,guanosine-monophosphate and cytidine-monophosphate monomers which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificsuccession of the monomers is called the RNA-sequence. Usually RNA maybe obtainable by transcription of a DNA-sequence, e.g., inside a cell.In eukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. Typically, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino-acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap, a5′-UTR, a coding region, a 3′-UTR and a poly(A) sequence. Aside frommessenger RNA, several non-coding types of RNA exist which may beinvolved in regulation of transcription and/or translation.

Stabilized nucleic acid, preferably mRNA: A stabilized nucleic acid,preferably mRNA typically, exhibits a modification increasing resistanceto in vivo degradation (e.g. degradation by an exo- or endo-nuclease)and/or ex vivo degradation (e.g. by the manufacturing process prior tovaccine administration, e.g. in the course of the preparation of thevaccine solution to be administered). Stabilization of RNA can, e.g., beachieved by providing a 5′-cap-Structure, a Poly-A-Tail, or any otherUTR-modification. It can also be achieved by chemical modification ormodification of the G/C content of the nucleic acid or other types ofsequence optimization. Various other methods are known in the art andconceivable in the context of the invention.

Sequence identity: Two or more sequences are identical if they exhibitthe same length and order of nucleotides or amino acids. The percentageof identity typically describes the extent, to which two sequences areidentical, i.e. it typically describes the percentage of nucleotidesthat correspond in their sequence position with identical nucleotides ofa reference sequence. In order to determine the degree of identity, thesequences to be compared are considered to exhibit the same length, i.e.the length of the longest sequence of the sequences to be compared. Thismeans that a first sequence consisting of 8 nucleotides is 80% identicalto a second sequence consisting of 10 nucleotides comprising the firstsequence. Hence, in the context of the present invention, identity ofsequences preferably relates to the percentage of nucleotides of asequence which have the same position in two or more sequences havingthe same length. Therefore, e.g. a position of a first sequence may becompared with the corresponding position of the second sequence. If aposition in the first sequence is occupied by the same component(residue) as is the case at a position in the second sequence, the twosequences are identical at this position. If this is not the case, thesequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul etal. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm isintegrated in the BLAST program. Sequences which are identical to thesequences of the present invention to a certain extent can be identifiedby this program.

Serotype, serotype of a virus: A serotype or a serotype of a virus is agroup of viruses classified together based on their antigens on thesurface of the virus, allowing the epidemiologic classification oforganisms to the sub-species level.

Strain, strain of a virus: A strain or a strain of a virus is a group ofviruses that are genetically distinct from other groups of the samespecies. The strain that is defined by a genetic variant is also definedas a “subtype”.

Transfection: The term “transfection” refers to the introduction ofnucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, intocells, preferably into eukaryotic cells. In the context of the presentinvention, the term “transfection” encompasses any method known to theskilled person for introducing nucleic acid molecules into cells,preferably into eukaryotic cells, such as into mammalian cells. Suchmethods encompass, for example, electroporation, lipofection, e.g. basedon cationic lipids and/or liposomes, calcium phosphate precipitation,nanoparticle based transfection, virus based transfection, ortransfection based on cationic polymers, such as DEAE-dextran orpolyethylenimine etc. Preferably, the introduction of the nucleic acid,preferably the mRNA is non-viral.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen, preferably animmunogen. The antigen or immunogen may be derived from any materialthat is suitable for vaccination. For example, the antigen or immunogenmay be derived from a pathogen, such as from bacteria or virus particlesetc., or from a tumor or cancerous tissue. The antigen or immunogenstimulates the body's adaptive immune system to provide an adaptiveimmune response.

Vector: The term “vector” refers to a nucleic acid molecule, preferablyto an artificial nucleic acid molecule. A vector in the context of thepresent invention is suitable for incorporating or harboring a desirednucleic acid sequence, such as a nucleic acid sequence comprising acoding region. Such vectors may be storage vectors, expression vectors,cloning vectors, transfer vectors etc. A storage vector is a vectorwhich allows the convenient storage of a nucleic acid molecule, forexample, of an mRNA molecule. Thus, the vector may comprise a sequencecorresponding, e.g., to a desired mRNA sequence or a part thereof, suchas a sequence corresponding to the coding region and the 3′-UTR and/orthe 5′-UTR of an mRNA. An expression vector may be used for productionof expression products such as RNA, e.g. mRNA, or peptides, polypeptidesor proteins. For example, an expression vector may comprise sequencesneeded for transcription of a sequence stretch of the vector, such as apromoter sequence, e.g. an RNA polymerase promoter sequence. A cloningvector is typically a vector that contains a cloning site, which may beused to incorporate nucleic acid sequences into the vector. A cloningvector may be, e.g., a plasmid vector or a bacteriophage vector. Atransfer vector may be a vector which is suitable for transferringnucleic acid molecules into cells or organisms, for example, viralvectors. A vector in the context of the present invention may be, e.g.,an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule.Preferably, a vector in the sense of the present application comprises acloning site, a selection marker, such as an antibiotic resistancefactor, and a sequence suitable for multiplication of the vector, suchas an origin of replication. Preferably, a vector in the context of thepresent application is a plasmid vector.

RNA in vitro transcription: The terms “RNA in vitro transcription” or“in vitro transcription” relate to a process wherein RNA is synthesizedin a cell-free system (in vitro). DNA, particularly plasmid DNA, is usedas template for the generation of RNA transcripts. RNA may be obtainedby DNA-dependent in vitro transcription of an appropriate DNA template,which according to the present invention is preferably a linearizedplasmid DNA template. The promoter for controlling in vitrotranscription can be any promoter for any DNA-dependent RNA polymerase.Particular examples of DNA-dependent RNA polymerases are the T7, T3, andSP6 RNA polymerases. A DNA template for in vitro RNA transcription maybe obtained by cloning of a nucleic acid, in particular cDNAcorresponding to the respective RNA to be in vitro transcribed, andintroducing it into an appropriate vector for in vitro transcription,for example into plasmid DNA. In a preferred embodiment of the presentinvention the DNA template is linearized with a suitable restrictionenzyme, before it is transcribed in vitro. The cDNA may be obtained byreverse transcription of mRNA or chemical synthesis. Moreover, the DNAtemplate for in vitro RNA synthesis may also be obtained by genesynthesis.

Methods for in vitro transcription are known in the art (see, e.g.,Geall et al. (2013) Semin. Immunol. 25(2):152-159: Brunelle et al.(2013) Methods Enzymol. 530:101-14). Reagents used in said methodtypically include:

-   -   1) a linearized DNA template with a promoter sequence that has a        high binding affinity for its respective RNA polymerase such as        bacteriophage-encoded RNA polymerases;    -   2) ribonucleoside triphosphates (NTPs) for the four bases        (adenine, cytosine, guanine and uracil);    -   3) optionally a cap analogue as defined above (e.g.        m7G(5′)ppp(5′)G (m7G)), optionally, fraction of NTPs optimized        to the RNA sequence (according to WD/2015/188933);    -   4) a DNA-dependent RNA polymerase capable of binding to the        promoter sequence within the linearized DNA template (e.g. T7,        T3 or SP6 RNA polymerase);    -   5) optionally a ribonuclease (RNase) inhibitor to inactivate any        contaminating RNase:    -   6) optionally a pyrophosphatase to degrade pyrophosphate, which        may inhibit transcription:    -   7) MgCl2, which supplies Mg2+ ions as a co-factor for the        polymerase;    -   8) a buffer to maintain a suitable pH value, which can also        contain antioxidants (e.g. DTT), and/or polyamines such as        spermidine at optimal concentrations.

Vehicle: A vehicle is typically understood to be a material that issuitable for storing, transporting, and/or administering a compound,such as a pharmaceutically active compound. For example, it may be aphysiologically acceptable liquid which is suitable for storing,transporting, and/or administering a pharmaceutically active compound.

Different species/species: The term “species” defines a monophyleticgroup of viruses whose properties can be distinguished from those ofother species by multiple criteria (Adams et al., 2013. Arch Virol 158:2633-9). The reference to a “different” or “2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 18, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more different” GI, GII, GIII, GIV or GV, GI.1 or GII.4 Norovirus orNoroviruses as used in the present invention means that a Norovirus fromanother species, strain or serotype is used or that the properties ofthe Noroviruses which are used or utilized can be distinguished fromthose of the other Norovirus by multiple criteria.

3′-untranslated region (3′-UTR): Generally, the term “3′-UTR” refers toa part of the artificial nucleic acid molecule, which is located 3′(i.e. “downstream”) of a coding region and which is not translated intoprotein. Typically, a 3′-UTR is the part of an mRNA which is locatedbetween the protein coding region (coding region or coding sequence(CDS)) and the poly(A) sequence of the mRNA. In the context of theinvention, the term 3′-UTR may also comprise elements, which are notencoded in the template, from which an RNA is transcribed, but which areadded after transcription during maturation, e.g. a poly(A) sequence. A3′-UTR of the mRNA is not translated into an amino acid sequence. The3′-UTR sequence is generally encoded by the gene which is transcribedinto the respective mRNA during the gene expression process. The genomicsequence is first transcribed into pre-mature mRNA, which comprisesoptional introns. The pre-mature mRNA is then further processed intomature mRNA in a maturation process. This maturation process comprisesthe steps of 5′capping, splicing the pre-mature mRNA to excize optionalintrons and modifications of the 3′-end, such as polyadenylation of the3′-end of the pre-mature mRNA and optional endo-/or exonucleasecleavages etc. In the context of the present invention, a 3′-UTRcorresponds to the sequence of a mature mRNA which is located betweenthe stop codon of the protein coding region, preferably immediately 3′to the stop codon of the protein coding region, and the poly(A) sequenceof the mRNA. The term “corresponds to” means that the 3′-UTR sequencemay be an RNA sequence, such as in the mRNA sequence used for definingthe 3′-UTR sequence, or a DNA sequence which corresponds to such RNAsequence. In the context of the present invention, the term “a 3′-UTR ofa gene”, is the sequence which corresponds to the 3′-UTR of the maturemRNA derived from this gene, i.e. the mRNA obtained by transcription ofthe gene and maturation of the pre-mature mRNA. The term “3′-UTR of agene” encompasses the DNA sequence and the RNA sequence (both sense andantisense strand and both mature and immature) of the 3′-UTR.Preferably, the 3′-UTRs have a length of more than 20, 30, 40 or 50nucleotides.

5′-untranslated region (5′-UTR): Generally, the term “5′-UTR” refers toa part of the artificial nucleic acid molecule, which is located 5′(i.e. “upstream”) of a coding region and which is not translated intoprotein. A 5′-UTR is typically understood to be a particular section ofmessenger RNA (mRNA), which is located 5′ of the coding region of themRNA. Typically, the 5′-UTR starts with the transcriptional start siteand ends one nucleotide before the start codon of the coding region.Preferably, the 5′-UTRs have a length of more than 20, 30, 40 or 50nucleotides. The 5′-UTR may comprise elements for controlling geneexpression, also called regulatory elements. Such regulatory elementsmay be, for example, ribosomal binding sites. The 5′-UTR may be posttranscriptionally modified, for example by addition of a 5′-CAP. A5′-UTR of the mRNA is not translated into an amino acid sequence. The5′-UTR sequence is generally encoded by the gene which is transcribedinto the respective mRNA during the gene expression process. The genomicsequence is first transcribed into pre-mature mRNA, which comprisesoptional introns. The pre-mature mRNA is then further processed intomature mRNA in a maturation process. This maturation process comprisesthe steps of 5′capping, splicing the pre-mature mRNA to excize optionalintrons and modifications of the 3′-end, such as polyadenylation of the3′-end of the pre-mature mRNA and optional endo-/or exonucleasecleavages etc. In the context of the present invention, a 5′-UTRcorresponds to the sequence of a mature mRNA which is located betweenthe start codon and, for example, the 5′-cap. Preferably, the 5′-UTRcorresponds to the sequence which extends from a nucleotide located 3′to the 5′-cap, more preferably from the nucleotide located immediately3′ to the 5′-cap, to a nucleotide located 5′ to the start codon of theprotein coding region, preferably to the nucleotide located immediately5′ to the start codon of the protein coding region. The nucleotidelocated immediately 3′ to the 5′-cap of a mature mRNA typicallycorresponds to the transcriptional start site. The term “corresponds to”means that the 5′-UTR sequence may be an RNA sequence, such as in themRNA sequence used for defining the 5′-UTR sequence, or a DNA sequencewhich corresponds to such RNA sequence. In the context of the presentinvention, the term “a 5′-UTR of a gene” is the sequence whichcorresponds to the 5′-UTR of the mature mRNA derived from this gene,i.e. the mRNA obtained by transcription of the gene and maturation ofthe pre-mature mRNA. The term “5′-UTR of a gene” encompasses the DNAsequence and the RNA sequence (both sense and antisense strand and bothmature and immature) of the 5′-UTR.

5′-Terminal Oligopyrimidine Tract (TOP): The 5′-terminal oligopyrimidinetract (TOP) is typically a stretch of pyrimidine nucleotides located inthe 5′-terminal region of a nucleic acid molecule, such as the5′-terminal region of certain mRNA molecules or the 5′-terminal regionof a functional entity, e.g. the transcribed region, of certain genes.The sequence starts with a cytidine, which usually corresponds to thetranscriptional start site, and is followed by a stretch of usuallyabout 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidinestretch and thus the 5′ TOP ends one nucleotide 5′ to the first purinenucleotide located downstream of the TDP. Messenger RNA that contains a5′-terminal oligopyrimidine tract is often referred to as TOP mRNA.Accordingly, genes that provide such messenger RNAs are referred to asTOP genes. TOP sequences have, for example, been found in genes andmRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is anucleic acid sequence which corresponds to a 5′TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TDP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′-end with a cytosine nucleotide. In TDP genes and TOPmRNAs, the TDP-motif preferably starts at its 5′-end with thetranscriptional start site and ends one nucleotide 5′ to the first purinresidue in said gene or mRNA. A TOP motif in the sense of the presentinvention is preferably located at the 5′-end of a sequence whichrepresents a 5′-UTR or at the 5′-end of a sequence which codes for a5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotidesis called “TOP motif” in the sense of the present invention if thisstretch is located at the 5′-end of a respective sequence, such as theartificial nucleic acid molecule, the 5′-UTR element of the artificialnucleic acid molecule, or the nucleic acid sequence which is derivedfrom the 5′-UTR of a TOP gene as described herein. In other words, astretch of 3 or more pyrimidine nucleotides, which is not located at the5′-end of a 5′-UTR or a 5′-UTR element but anywhere within a 5′-UTR or a5′-UTR element, is preferably not referred to as “TOP motif”.

TOP gene: TOP genes are typically characterised by the presence of a5′-terminal oligopyrimidine tract. Furthermore, most TOP genes arecharacterized by a growth-associated translational regulation. However,also TOP genes with a tissue specific translational regulation areknown. As defined above, the 5′-UTR of a TOP gene corresponds to thesequence of a 5′-UTR of a mature mRNA derived from a TOP gene, whichpreferably extends from the nucleotide located 3′ to the 5′-cap to thenucleotide located 5′ to the start codon. A 5′-UTR of a TOP genetypically does not comprise any start codons, preferably no upstreamAUGs (uAUGs) or upstream coding regions (uORFs). Therein, upstream AUGsand upstream coding regions are typically understood to be AUGs andcoding regions that occur 5′ of the start codon (AUG) of the codingregion that should be translated. The 5′-UTRs of TOP genes are generallyrather short. The lengths of 5′-UTRs of TOP genes may vary between 20nucleotides up to 500 nucleotides, and are typically less than about 200nucleotides, preferably less than about 150 nucleotides, more preferablyless than about 100 nucleotides. Exemplary 5′-UTRs of TOP genes in thesense of the present invention are the nucleic acid sequences extendingfrom the nucleotide at position 5 to the nucleotide located immediately5′ to the start codon (e.g. the ATG) in the sequences according to SEQID NOs: 1-1383 of the patent application WO 2013/143700, whosedisclosure is incorporated herewith by reference. In this context aparticularly preferred fragment of a 5′-UTR of a TOP gene is a 5′-UTR ofa TOP gene lacking the 5′TOP motif. The terms “5′-UTR of a TOP gene” or“5′-TOP UTR” preferably refer to the 5′-UTR of a naturally occurring TOPgene.

Orthologues and paralogues: Orthologues and paralogues encompassevolutionary concepts used to describe the ancestral relationships ofgenes. Paralogues are genes within the same species that have originatedthrough duplication of an ancestral gene; orthologues are genes fromdifferent organisms that have originated through speciation, and arealso derived from a common ancestral gene. In the context of theinvention, an orthologue and/or a paralogue of a Norovirus nucleic acidsequence of the invention preferably refers to a sequence having inincreasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58% 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity to the sequence as represented bySEQ ID NOs: 4411-39690, 39713-39748. In the context of the invention, anorthologue and/or a paralogue of a Norovirus amino acid sequence of theinvention refers preferably to a sequence having in increasing order ofpreference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 68%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moresequence identity to the sequence as represented by SEQ ID NOs: 1-4410.

Hybridization/Homology: Nucleic acid molecules which are advantageouslyfor the process according to the invention can be isolated based ontheir homology to the nucleic acid molecules or a complement sequence ofthe nucleic acid molecules disclosed herein using the sequences or partthereof as hybridization probe and following standard hybridizationtechniques under stringent hybridization conditions. In this context, itis possible to use, for example, isolated nucleic acid molecules of atleast 15, 20, 25, 30, 35, 40, 50, 60 or more nucleotides, preferably ofat least 15, 20 or 25 nucleotides in length which hybridize understringent conditions with the above-described nucleic acid molecules, inparticular with those which encompass a nucleotide sequence of thenucleic acid molecule used in the invention or encoding a protein usedin the invention or of the nucleic acid molecule of the invention.Nucleic acid molecules with 35, 50, 100, 250 or more nucleotides mayalso be used.

The term “homology” means that the respective nucleic acid molecules orencoded proteins are functionally and/or structurally equivalent. Thenucleic acid molecules that are homologous to the nucleic acid moleculesdescribed above and that are derivatives of said nucleic acid moleculesare, for example, variations of said nucleic acid molecules whichrepresent modifications having the same biological function, inparticular encoding proteins with the same or substantially the samebiological function. They may be naturally occurring variations, such assequences from other species, strains, or mutations. These mutations mayoccur naturally or may be obtained by mutagenesis techniques. Theallelic variations may be naturally occurring allelic variants as wellas synthetically produced or genetically engineered variants.Structurally equivalents can, for example, be identified by testing thebinding of said polypeptide to antibodies or computer based predictions.

By “hybridizing” it is meant that such nucleic acid molecules hybridizeunder conventional hybridization conditions, preferably under stringentconditions such as described by, e.g., Sambrook (Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989)) or in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

According to the invention, DNA as well as RNA molecules of the nucleicacid of the invention can be used as probes. Further, as template forthe identification of functional homologues Northern blot assays as wellas Southern blot assays can be performed. The Northern blot assayadvantageously provides further information about the expressed geneproduct: e.g. expression pattern, occurrence of processing steps, likesplicing and capping, etc. The Southern blot assay provides additionalinformation about the chromosomal localization and organization of thegene encoding the nucleic acid molecule of the invention.

A preferred, no limiting example of stringent hydridization conditionsare hybridizations in 6×sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more wash steps in 1.2×SSC,0.1% SDS at 50 to 65° C., for example at 50° C., 55° C. or 60° C. Theskilled worker knows that these hybridization conditions differ as afunction of the type of the nucleic acid and, for example when organicsolvents are present, with regard to the temperature and concentrationof the buffer. The temperature under “standard hybridization conditions”differs for example as a function of the type of the nucleic acidbetween 42° C. and 58° C., preferably between 45° C. and 50° C. in anaqueous buffer with a concentration of 0.1×0.5×, 1×, 2×, 3×, 4× or 5×SSC(pH 7.2). If organic solvent(s) is/are present in the abovementionedbuffer, for example 50% formamide, the temperature under standardconditions is approximately 40° C., 42° C. or 45° C. The hybridizationconditions for DNA:DNA hybrids are preferably for example 0.1×SSC and20° C., 25° C., 30° C., 35° C., 40° C. or 45° C., preferably between 30°C. and 45° C. The hybridization conditions for DNA:RNA hybrids arepreferably for example 0.1×SSC and 30° C., 35° C. 40° C., 45° C., 50° C.or 55° C., preferably between 45° C. and 55° C. The abovementionedhybridization temperatures are determined for example for a nucleic acidapproximately 101 bp (=base pairs) in length and a G+C content of 50% inthe absence of formamide. The skilled worker knows to determine thehybridization conditions required with the aid of textbooks, for examplethe ones mentioned above, or from the following textbooks: Sambrook etal., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames andHiggins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”,IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991,“Essential Molecular Biology: A Practical Approach”, IRL Press at OxfordUniversity Press, Oxford.

A further example of one such stringent hybridization condition ishybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for one hour. Alternatively, an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Further, the conditionsduring the wash step can be selected from the range of conditionsdelimited by low-stringency conditions (approximately 2×SSC at 50° C.)and high-stringency conditions (approximately 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Inaddition, the temperature during the wash step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tohigher-stringency conditions at approximately 65° C. Both of theparameters salt concentration and temperature can be variedsimultaneously, or else one of the two parameters can be kept constantwhile only the other is varied. Denaturants, for example formamide orSOS, may also be employed during the hybridization. In the presence of50% formamide, hybridization is preferably effected at 42° C. Relevantfactors like i) length of treatment, ii) salt conditions, iii) detergentconditions, iv) competitor DNAs, v) temperature and vi) probe selectioncan be combined case by case so that not all possibilities can bementioned herein.

Some examples of conditions for DNA hybridization (Southern blot assays)and wash step are shown herein below:

-   -   (1) Hybridization conditions can be selected, for example, from        the following conditions:        -   a) 4×SSC at 65° C.,        -   b) 6×SSC at 45° C.,        -   c) 6×SSC, 100 mg/ml denatured fragmented fish sperm DNA at            68° C.,        -   d) 6×SSC, 0.5% SOS, 100 mg/ml denatured salmon sperm DNA at            68° C.,        -   e) 6×SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon            sperm DNA, 50% formamide at 42° C.,        -   f) 50% formamide, 4×SSC at 42° C.,        -   g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1%            Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate            buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42° C.,        -   h) 2× or 4×SSC at 50° C. (low-stringency condition), or        -   i) 30 to 40% formamide, 2× or 4×SSC at 42° C.            (low-stringency condition).    -   (2) Wash steps can be selected, for example, from the following        conditions:        -   a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SOS at 50° C.        -   b) 0.1×SSC at 65° C.        -   c) 0.1×SSC, 0.5% SDS at 68° C.        -   d) 0.1×SSC, 0.5% SOS, 50% formamide at 42° C.        -   e) 0.2×SSC, 0.1% SDS at 42° C.        -   f) 2×SSC at 65° C. (low-stringency condition).

Further, some applications have to be performed at low stringencyhybridisation conditions, without any consequences for the specificityof the hybridisation. For example, a Southern blot analysis of total DNAcould be probed with a nucleic acid molecule of the present inventionand washed at low stringency (55° C. in 2×SSPE, 0.1% SDS). A furtherexample of such low-stringent hybridization conditions is 4×SSC at 50°C. or hybridization with 30 to 40% formamide at 42° C. Such moleculescomprise those which are fragments, analogues or derivatives of thepolypeptide of the invention or used in the methods of the invention anddiffer, for example, by way of amino acid and/or nucleotide deletion(s),insertion(s), substitution (s), addition(s) and/or recombination (s) orany other modification(s) known in the art either alone or incombination from the above-described amino acid sequences or theirunderlying nucleotide sequence(s). However, it is preferred to use highstringency hybridisation conditions.

Hybridization should advantageously be carried out with fragments of atleast 5, 10, 15, 20, 25, 30, 35 or 40 bp, advantageously at least 50,60, 70 or 80 bp, preferably at least 90, 100 or 110 bp. Most preferablyare fragments of at least 15, 20, 25 or 30 bp. Preferably are alsohybridizations with at least 100 bp or 200, very especially preferablyat least 400 bp in length. In an especially preferred embodiment, thehybridization should be carried out with the entire nucleic acidsequence with conditions described above.

The term “hybridizes under stringent conditions” is defined above. Inone embodiment, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 30%, 40%, 50% or 65% identical toeach other typically remain hybridized to each other. Preferably, theconditions are such that sequences at least about 70%, more preferablyat least about 75% or 80%, and even more preferably at least about 85%,90% or 95% or more identical to each other typically remain hybridizedto each other.

To determine the percentage homology (=identity, herein usedinterchangeably) of two amino acid sequences or of two nucleic acidmolecules, the sequences are written one underneath the other for anoptimal comparison (for example gaps may be inserted into the sequenceof a protein or of a nucleic acid in order to generate an optimalalignment with the other protein or the other nucleic acid).

The amino acid residues or nucleic acid molecules at the correspondingamino acid positions or nucleotide positions are then compared. If aposition in one sequence is occupied by the same amino acid residue orthe same nucleic acid molecule as the corresponding position in theother sequence, the molecules are homologous at this position (i.e.amino acid or nucleic acid “homology” as used in the present contextcorresponds to amino acid or nucleic acid “identity”. The percentagehomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e. % homology=number ofidentical positions/total number of positions×100). The terms “homology”and “identity” are thus to be considered as synonyms.

For the determination of the percentage homology (=identity) of two ormore amino acids or of two or more nucleotide sequences several computersoftware programs have been developed. The homology of two or moresequences can be calculated with for example the software fasta, whichpresently has been used in the version fasta 3 (W. R. Pearson and D. J.Lipman (1988), Improved Tools for Biological Sequence Comparison. PNAS85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive SequenceComparison with FASTP and FASTA, Methods in Enzymology 183:63-98: W. R.Pearson and D. J. Lipman (1988) Improved Tools for Biological SequenceComparison. PNAS 85:2444-2448: W. R. Pearson (1990); Rapid and SensitiveSequence Comparison with FASTP and FASTAMethods in Enzymology183:83-98). Another useful program for the calculation of homologies ofdifferent sequences is the standard blast program, which is included inthe Biomax pedant software (Biomax, Munich, Federal Republic ofGermany). This leads unfortunately sometimes to suboptimal results sinceblast does not always include complete sequences of the subject and thequery. Nevertheless as this program is very efficient it can be used forthe comparison of a huge number of sequences. The following settings aretypically used for such a comparisons of sequences: -p Program Name[String]; -d Database [String]; default=nr; -i Query File [File In];default=stdin; -e Expectation value (E) [Real]; default=10.0; -malignment view options: 0=pairwise; 1=query-anchored showing identities;2=query-anchored no identities; 3=flat query-anchored, show identities;4=flat query-anchored, no identities; 5=query-anchored no identities andblunt ends; 6=flat query-anchored, no identities and blunt ends; 7=XMLBlast output: 8=tabular; 9 tabular with comment lines [Integer]:default=0; -o BLAST report Output File [File Out] Optional;default=stdout; -F Filter query sequence (DUST with blastn, SEB withothers) [String]; default=T; -G Cost to open a gap (zero invokes defaultbehavior) [Integer]; default=D; -E Cost to extend a gap (zero invokesdefault behavior) [Integer]: default=0: -X X drop-off value for gappedalignment (in bits) (zero invokes default behavior); blastn 30, megablast 21, tblastx 0, all others 15 [Integer]; default=0; -l Show GI's indeflines [T/F]; default=F; -q Penalty for a nucleotide mismatch (blastnonly) [Integer]; default=−3; -r Reward for a nucleotide match (blastnonly) [Integer]: default=1; -v Number of database sequences to showone-line descriptions for (V) [Integer]; default=500; -b Number ofdatabase sequence to show alignments for (B) [Integer]; default=250; -fThreshold for extending hits, default if zero; blastp 11, blastn 0,blastx 12, tblastn 13; tblastx 13, mega blast 0 [Integer]; default=0; -gPerfom gapped alignment (not available with tblastx) [T/F]; default=T;-Q Query Genetic code to use [Integer]; default=1; -D DB Genetic code(for tblast[nx] only) [Integer]; default=1; -a Number of processors touse [Integer]; default=; -O SeqAlign file [File Out] Optional; -JBelieve the query defline [T/F]; default=F; -M Matrix [String];default=BLOSUM62; -W Word size, default if zero (blastn II, megablast28, all others 3) [Integer]; default=0; -z Effective length of thedatabase (use zero for the real size) [Real]; default=0; -K Number ofbest hits from a region to keep (off by default, if used a value of 100is recommended) [Integer]; default=0; -P 0 for multiple hit, 1 forsingle hit [Integer]; default=0; -Y Effective length of the search space(use zero for the real size) [Real]; default=0; -S Query strands tosearch against database (for blast[nx], and tblastx); 3 is both, 1 istop, 2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];default=F: -I Restrict search of database to list of GI's [String]Optional; -U Use lower case filtering of FASTA sequence [T/F] Optional;default=F; -y X dropoff value for ungapped extensions in bits (0.0invokes default behavior); blastn 20, megablast 10, all others 7 [Real];default=0.0: -Z X dropoff value for final gapped alignment in bits (0.0invokes default behavior); blastn/megablast 50, tblastx 0, all others 25[Integer]; default=0; -R PSI-TBLASTN checkpoint file [File In] Optional;-n MegaBlast search [T/F]; default=F; -L Location on query sequence[String] Optional; -A Multiple Hits window size, default if zero(blastn/megablast 0, all others 40 [Integer]; default=0: -w Frame shiftpenalty (00F algorithm for blastx) [Integer]; default=0; -t Length ofthe largest intron allowed in tblastn for linking HSPs (0 disableslinking) [Integer]; default=0.

Results of high quality are reached by using the algorithm of Needlemanand Wunsch or Smith and Waterman. Therefore programs based on saidalgorithms are preferred. Advantageously the comparisons of sequencescan be done with the program PileUp (J. Mol. Evolution., 25, 351 (1987),Higgins et al., CABIOS 5, 151 (1989)) or preferably with the programs“Gap” and “Needle”, which are both based on the algorithms of Needlemanand Wunsch (J. Mol. Biol. 48; 443 (1970)), and “BestFit”, which is basedon the algorithm of Smith and Waterman (Adv. Appl. Math. 2; 482 (1981)).“Gap” and “BestFit” are part of the GCG software-package (GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991);Altschul et al., (Nucleic Acids Res. 25, 3389 (1997)), “Needle” is partof the European Molecular Biology Open Software Suite (EMBOSS) (Trendsin Genetics 16 (B), 276 (2000)). Therefore preferably the calculationsto determine the percentages of sequence homology are done with theprograms “Gap” or “Needle” over the whole range of the sequences. Thefollowing standard adjustments for the comparison of nucleic acidsequences were used for “Needle”: matrix: EDNAFULL, Gap_penalty: 10.0.Extend_penalty: 0.5. The following standard adjustments for thecomparison of nucleic acid sequences were used for “Gap”: gap weight:50, length weight: 3, average match: 10.000, average mismatch: 0.000.

For example a sequence, which has 80% homology with sequence SEQ ID NO:4411 at the nucleic acid level is understood as meaning a sequencewhich, upon comparison with the sequence SEQ ID NO: 4411 by the aboveprogram “Needle” with the above parameter set, has a 80% identity.

Homology between two polypeptides is understood as meaning the identityof the amino acid sequence over in each case the entire sequence lengthwhich is calculated by comparison with the aid of the above program“Needle” using Matrix: EBLOSUM62, Gap_penalty: 8.0, Extend_penalty: 2.0.

For example a sequence which has a 80% homology with sequence SEQ ID NO:1 at the protein level is understood as meaning a sequence which, uponcomparison with the sequence SEQ ID NO: 1 by the above program “Needle”with the above parameter set, has a 80% identity.

DETAILED DESCRIPTION

In a first aspect, the invention relates to an artificial nucleic acidcomprising or consisting of at least one coding region encoding at leastone polypeptide derived from a Norovirus, and/or a fragment or variantthereof. In another embodiment, the artificial nucleic acid comprises atleast one coding region encoding at least one polypeptide comprising orconsisting of at least one Norovirus capsid protein, and/or a fragmentor variant thereof.

In one embodiment, the artificial nucleic acid molecule according to theinvention comprises at least one coding region encoding the Noroviruscapsid protein VP1 or VP2. In another embodiment, the artificial nucleicacid encodes Norovirus capsid protein VP1. In this context, a referenceto a Norovirus capsid protein VP1 equals a reference to a VP1 capsidprotein derived from a Norovirus, a Norovirus VP1, capsid protein,capsid protein VP1, major capsid protein, major capsid protein VP1,major capsid region, major viral capsid protein, or VP1 capsid protein.

In particular, the invention relates to an artificial nucleic acidcomprising or consisting of at least one coding region encoding at leastone polypeptide selected from the group consisting of Norovirus capsidprotein VP1 (also termed “Major capsid protein” or “capsidprotein”),Norovirus capsid protein VP2 (also termed “Minor capsid protein”) and/ora Norovirus non-structural protein, such as NS1/NS2 (also termed p48 orNterm (amino terminal protein)), NS3 (also termed NTPase or Nucleosidetriphosphatase), NS4 (also termed p22 or 3A-like protein), NS5 (alsotermed VP1 or Genome-linked viral protein), NS6 (also termed Pro orProteinase), or NS7 (also termed Pol or RNA-dependent RNA polymerase),and/or a fragment or variant of any of these proteins.

In one embodiment of the invention, vaccines and/or compositions containVP1 proteins and/or VP2 proteins. Preferably each vaccine and/orcomposition contains VP1 and/or VP2 proteins from only one Norovirusgenogroup giving rise to a monovalent vaccine. As used herein, the term“monovalent” means the antigenic proteins are derived from a singleNorovirus genogroup. For example, the vaccines and/or compositionscontain VP1 and/or VP2 from a virus strain of genogroup I (e.g. VP1 andVP2 from Norwalk virus).

Preferably the vaccines and/or compositions are comprised ofpredominantly VP1 proteins. In one embodiment of the invention, theantigen is a mixture of monovalent vaccines and/or compositions whereinthe composition includes vaccines and/or compositions comprised of VP1and/or VP2 from a single Norovirus genogroup mixed with vaccines and/orcompositions comprised of VP1 and/or VP2 from a different Norovirusgenogroup taken from multiple viral strains (e.g. a Norovirus fromGenogroup IV.2 and a Norovirus from Genogroup I.1). Purely by way ofexample the composition can contain monovalent vaccines and/orcompositions from one or more strains of Norovirus genogroup I togetherwith monovalent vaccines and/or compositions from one or more strains ofNorovirus genogroup II. Preferably, the Norovirus vaccines and/orcomposition mixture is composed of the Norovirus from Genogroup IV.2 andNorovirus from Genogroup I.1 or from different genus or species of aNorovirus from Genogroup IV.2.

Preferably the vaccines and/or compositions are comprised ofpredominantly VP1 proteins. In one embodiment of the invention, theantigen is a mixture of monovalent vaccines and/or compositions whereinthe composition includes vaccines and/or compositions comprised of VP1and/or VP2 from a single Norovirus genogroup mixed with vaccines and/orcompositions comprised of VP1 and/or VP2 from a different Norovirusgenogroup taken from multiple viral strains (e.g. a Norovirus fromGenogroup IV.2 and a Norovirus from genogroup I.1). Purely by way ofexample the composition can contain monovalent vaccines and/orcompositions from one or more strains of Norovirus genogroup I togetherwith monovalent vaccines and/or compositions from one or more strains ofNorovirus genogroup II. Preferably, the Norovirus vaccines and/orcomposition mixture is composed of the Norovirus from Genogroup II.4 andNorovirus from Genogroup I.1 or from different genus or species of aNorovirus from Genogroup II.4.

In this context, the amino acid sequence of the at least one antigenicpeptide or protein may be selected from any peptide or protein derivedfrom a capsid protein VP1, capsid protein VP2, NS1/NS2, NS3, NS4, NS5,NS6, or NS7 of a Norovirus or a fragment or variant thereof.

Further, in an alternative embodiment of the invention, the vaccinesand/or compositions may be multivalent vaccines and/or compositions thatcomprise, for example, VP1 and/or VP2 proteins from one Norovirusgenogroup intermixed with VP1 and/or VP2 proteins from a secondNorovirus genogroup, wherein the different VP1 and VP2 proteins are notchimeric VP1 and VP2 proteins, but associate together within the samecapsid structure to form immunogenic Vaccines and/or compositions. Asused herein, the term “multivalent” means that the antigenic proteinsare derived from two or more Norovirus genogroups. Multivalent vaccinesand/or compositions may contain vaccines and/or composition antigenstaken from two or more viral strains. Purely by way of example thecomposition can contain multivalent vaccines and/or compositionscomprised of capsid monomers or multimers from one or more strains ofNorovirus genogroup I together with capsid monomers or multimers fromone or more strains of Norovirus genogroup II. Preferably, the Norovirusvaccines and/or composition mixture is composed of the Norovirus fromGenogroup IV.2 and Norovirus from Genogroup I.1 or from different genusor species of a Norovirus from Genogroup IV.2.

Further, in an alternative embodiment of the invention, the vaccinesand/or compositions may be multivalent vaccines and/or compositions thatcomprise, for example, VP1 and/or VP2 proteins from one Norovirusgenogroup intermixed with VP1 and/or VP2 proteins from a secondNorovirus genogroup, wherein the different VP1 and VP2 proteins are notchimeric VP1 and VP2 proteins, but associate together within the samecapsid structure to form immunogenic Vaccines and/or compositions. Asused herein, the term “multivalent” means that the antigenic proteinsare derived from two or more Norovirus genogroups. Multivalent vaccinesand/or compositions may contain vaccines and/or composition antigenstaken from two or more viral strains. Purely by way of example thecomposition can contain multivalent vaccines and/or compositionscomprised of capsid monomers or multimers from one or more strains ofNorovirus genogroup I together with capsid monomers or multimers fromone or more strains of Norovirus genogroup II. Preferably, the Norovirusvaccines and/or composition mixture is composed of the Norovirus fromGenogroup II.4 and Norovirus from Genogroup I.1 or from different genusor species of a Norovirus from Genogroup II.4.

In another embodiment, the artificial nucleic acid may comprise 2, 3, 4,5, 6, 7, 8, 9, 10 or more coding regions encoding at least onepolypeptide selected from the group consisting of Norovirus capsidprotein VP1, Norovirus capsid protein VP2, NS1/NS2, NS3, NS4, NS5, NS6,or NS7, and/or a fragment or variant of any of these proteins.

In a preferred embodiment, the compositions or vaccines of the presentinvention comprise a multivalent vaccine, e.g., comprising apolynucleotide which encodes at least two different VP1, for example aNorovirus capsid protein VP1 from a GII.4 strain and a Norovirus capsidprotein VP1 from a GI.1 strain. In another embodiment, the compositionsor vaccines comprises a multivalent vaccine, e.g., comprising twodifferent polynucleotides, whereby one polynucleotide encodes forexample a Norovirus capsid protein VP1 from a GII.4 strain and the otherpolynucleotide encodes a Norovirus capsid protein VP1 from a GI.1strain.

In a further embodiment, the compositions or vaccines of the presentinvention may comprise a multivalent vaccine, e.g., comprising apolynucleotide which encodes at least two different VP1, for example aNorovirus capsid protein VP1 from two different GII.4 strains. Inanother embodiment, the compositions or vaccines comprise a multivalentvaccine, e.g., comprising two different polynucleotides, whereby onepolynucleotide encodes for example a Norovirus capsid protein VP1 fromtwo different GII.4 strains.

In some embodiments, as defined above, the composition or the vaccine ismultivalent, i.e. compositions or vaccines of the present invention mayvary in their valency. Valency refers to the number of antigeniccomponents in the composition or vaccine. In some embodiments, thecompositions or vaccines are monovalent. In some embodiments, thecompositions or vaccines are divalent or bivalent. In some embodimentsthe compositions or vaccines are trivalent. In some embodiments thecompositions or vaccines are teravalent. In some embodiments thecompositions or vaccines are multi-valent. Multivalent vaccines maycomprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 38, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 7, 68, 89, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more antigens orantigenic moieties (e.g., antigenic peptides, etc.). The antigeniccomponents of the compositions or vaccines may be on a singlepolynucleotide or on separate polynucleotides. In another embodiment,multivalent vaccines may comprise or at least 10, 15, 20, 30, 40 or 50or 100 or more antigens or antigenic moieties (e.g., antigenic peptides,etc.). In another embodiment, multivalent vaccines may comprise 2-10,10-15, 15-20, 20-50, 50-100 or 100-200 or more antigens or antigenicmoieties (e.g., antigenic peptides, etc.).

In a preferred embodiment, the multivalent composition or vaccinecomprises about 30 to about 50 antigens or antigenic moieties.

In some embodiments, the open reading frame of the one or more RNApolynucleotides encodes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or atleast 10, 15, 20 or 50 or 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200antigenic polypeptides.

In this context it is particularly preferred that the compositioncomprises at least two mRNA sequences, wherein at least one mRNAsequence encodes at least one antigenic peptide or protein, i.e. VP1, isderived from a GII.4 Norovirus and at least one mRNA sequence encodes atleast one antigenic peptide or protein, derived from Norovirus VP1, isderived from another GII.4 Norovirus.

In another preferred embodiment each mRNA sequence encodes at least onedifferent antigenic peptide or protein derived from proteins ofdifferent Noroviruses. Preferably each mRNA sequence encodes at leastone antigenic peptide or protein, derived from Norovirus VP1, ofdifferent GII.4 Noroviruses or different GI.1 Noroviruses, or acombination thereof.

In a further embodiment, the invention relates to a compositioncomprising or consisting of 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more separateartificial nucleic acids selected from the group of SEQ ID NOs:4411-39890, 39713-39746 comprising at least one coding region encodingat least one polypeptide derived from a Norovirus selected from thegroup SEQ ID NOs: 1-4410 or a fragment or variant of any of theseproteins.

In some embodiments, the open reading frame of the one or more RNApolynucleotides encode at least 10, 15, 20, 30, 40 or 50 or 100antigenic polypeptides. In some embodiments, the open reading frame ofthe one or more RNA polynucleotides encode 2-10, 10-15, 15-20, 20-50,50-100 or 100-200 antigenic polypeptides.

The present invention is based on the surprising finding that the atleast one Norovirus protein comprised in the at least one polypeptideencoded by the artificial nucleic acid as described herein canefficiently be expressed in a mammalian cell. It was furtherunexpectedly found that the artificial nucleic acid is suitable foreliciting an immune response against Norovirus in a subject.

Furthermore, the present invention is based on the surprising findingthat mRNA-based or artificial nucleic acid vaccines comprising mRNA orartificial nucleic acid sequences encoding different antigens of aNorovirus (particularly Norovirus capsid protein VP1) were extremelyeffective in inducing an antigen-specific immune response againstNorovirus. Furthermore, the inventors surprisingly found that many mRNAsequences encoding different antigens of different Noroviruses can beeffectively combined in one mRNA-based vaccine.

In one embodiment, the artificial nucleic acid of the inventioncomprises at least one coding region encoding at least one polypeptidederived from a Norovirus and/or a fragment or variant thereof.

In a further embodiment, the artificial nucleic acid of the inventioncomprises at least one coding region encoding at least one polypeptideselected from the group consisting of a non-structural protein derivedfrom a Norovirus and/or a capsid protein derived from a Norovirus,and/or a fragment or variant thereof.

In a further embodiment, the artificial nucleic acid of the inventioncomprises at least one coding region encoding at least one polypeptideselected from the group consisting of Norovirus non-structural proteinsNS1/NS2, NS3, NS4, NS5, NS6, NS7, Norovirus capsid protein VP1 andNorovirus capsid protein VP2, and/or a fragment or variant thereof.

Norovirus:

In the context of the present invention, the term “Norovirus” comprisesany Norovirus, irrespective of strain or origin. Preferably, the term“Norovirus” comprises a Norovirus strain selected from the groupconsisting of Genogroup I, Genogroup II, Genogroup III, Genogroup IV, orGenogroup V (abbreviated as I, II, GIII, GIV or GV, respectively).

In a further embodiment, the term “Norovirus” comprises a Norovirusstrain selected from the group consisting of

-   -   (i) Genogroup I genotype 1 (abbreviated as GI.1), GI.2, GI.3,        GI.4, GI.5, GI.6, GI.7, GI.8, GI.9, GI.10, GI.11, GI.12, GI.13,        GI.14, GI.15. GI.16 and/or GI.17;    -   (ii) Genogroup II genotype 1 (abbreviated as GII.1), GII.2,        GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.9, GII.10, GII.11,        GII.12, GII.13, GII.14, GII.15, GII.16, GII.17, GII.18, GII.19,        GII.20, GII.21, GII.22, GII.23, and/or GII.24;    -   (iii) Genogroup III genotype 1 (abbreviated as GIII.1), GIII.2,        GIII.3, and/or GIII.4;    -   (iv) Genogroup IV genotype 1 (abbreviated as GIV.1), GIV.2,        GIV.3, and/or GIV.4;    -   (v) Genogroup V genotype 1 (abbreviated as GV.1), GV.2, GV.3,        and/or GV.4;    -   and/or combinations of 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more of        any of the above Noroviruses from different Genogroups and/or        different Genotypes.

In a further embodiment, the nucleic acid of the invention, preferably aVP1 nucleic acid derived from a Norovirus, is derived from a Norovirusselected from the group consisting of

-   -   (i) Genogroup I genotype 1 (abbreviated as GI.1). GI.2, GI.3,        GI.4, GI.5, GI.6, GI.7, GI.8, GI.9, GI.10, GI.11, GI.12, GI.13.        GI.14, GI.15, GI.16 and/or GI.17;    -   (ii) Genogroup II genotype 1 (abbreviated as GII.1), GII.2,        GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.9, GII.10, GII.11,        GII.12, GII.13, GII.14, GII.15, GII.16, GII.17, GII.18, GII.19,        GII.20, GII.21, GII.22, GII.23, and/or GII.24;    -   (iii) Genogroup III genotype 1 (abbreviated as GIII.1), GIII.2,        GIII.3, and/or GIII.4;    -   (iv) Genogroup IV genotype 1 (abbreviated as GIV.1). GIV.2,        GIV.3, and/or GIV.4; and    -   (v) Genogroup V genotype 1 (abbreviated as GV.1), GV.2, GV.3,        and/or GV.4.

In the case, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different VP1 nucleicacids are utilized or employed, for example in a composition of theinvention or a vaccine of the invention, the VP1 nucleic acids can bederived from 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more of any of the aboveNoroviruses from different Genogroups and/or different Genotypes.

In the context of the invention, a reference to GI.1 to GI.17 means areference to GI.1, GI.2, GI.3, GI.4, GI.5, GI.6, GI.7, GI.8, GI.9,GI.10, GI.11, GI.12, GI.13, GI.14, GI.15, GI.16 and/or GI.17; areference to GII.1 to GII.24 means a reference to GII.1, GII.2, GII.3,GII.4, GII.5, GII.6, GII.7, GII.8, GII.9, GII.10, GII.11, GII.12,GII.13, GII.14, GII.15, GII.16, GII.17, GII.18, GII.19, GII.20, GII.21,GII.22, GII.23, and/or GII.24; a reference to GIII.1 to GIII.4 means toa reference to GIII.1, GIII.2, GIII.3, and/or GIII.4; a reference toGIV.1 to GIV.4 means to a reference to GIV.1, GIV.2, GIV.3, and/orGIV.4; a reference to GV.1 to GV.4 means to a reference to GV.1, GV.2,GV.3, and/or GV.4.

In other embodiments, the term “Norovirus” comprises a Norovirus strainselected from the group consisting of a GII.4 Norovirus and/or a GI.1Norovirus. In a further embodiment, the term “Norovirus” as used hereinrefers to Norovirus GII.4 CIN-1 (also termed NorovirusHu/GII.4/031693/USA/2003 or having Accession No. JQ965810.1), GII.4(Accession No: AY502023.1), GII.4 CIN-002 and/or GII.4 Sydney. Inanother embodiment, the Norovirus is a GII.4 Sydney Norovirus or a GII.4Sydney 2012 Norovirus.

In other embodiments, the term “Norovirus” comprises a Norovirus strainselected from the group consisting of a GII.4 Norovirus and/or a GI.1Norovirus. In a further embodiment, the term “Norovirus” as used hereinrefers to Norovirus GII.4-031693-USA-2003, Norovirus GII.4/FarmingtonHills/2002/USA, Norovirus GII.4-2006b 092895-USA-2008, NorovirusGII.4-GZ2010-L87-Guangzhou-2011, Norovirus GII.4-USA-1997, NorovirusGI.1-USA-1968-Capsidprotein.

In another embodiment, the term “Norovirus” comprises a Norovirus strainselected from the group consisting of NorovirusHu/GII.4/Dijon/E872/2002/FRA, Norovirus Hu/GII.4/MD120-12/1987/USA,Norovirus Hu/GII.1/7EK/Hawaii/1971/USA, NorovirusHu/GII.6/CHDC4073/1984/USA, Norovirus Hu/GII.4/Hiroshima/19/2001/JPN,Norovirus Hu/GII.4/Hiroshima/97/2006/JPN, NorovirusHu/GII/JP/2015/GII.Pe_GII.4/Osaka/OSF78, NorovirusGII/Hu/NL/2012/GII.4/Groningen, NorovirusGII/Hu/NL/2014/GII.2/Groningen, Norovirus Hu/GII.4/NewOrleans1500/2008/USA, Norovirus Hu/GII.6/Ohio/490/2012/USA, NorovirusHu/GII.3/Jingzhou/2013402/CHN, Norovirus Hu/GII.4/Jingzhau/2013403/CHN,Norovirus Hu/GII.17/Gaithersburg/2014/US, NorovirusHu/GII.4/C127/GF/1978, Norovirus Hu/GII.4/CHDC3967/1988/03, NorovirusHu/GII.4/CHDC4108/1987/US, Norovirus Hu/GII.4/CHDC4871/1977/US,Norovirus Hu/GII.3/CHDC5291/1990/US, NorovirusHu/GII.3/Milwaukee009/2010/USA, NorovirusHu/GII.4/Miranda/NSW817L/2010/AU, Norovirus Hu/GII.2/KL109/MY/1978,Norovirus Hu/GII.14/HK74/CN/1978, Norovirus Hu/GII.7/HK4/CN/1976,Norovirus Hu/GII.17/C142/GF/1978, Norovirus Hu/GII.5/C15/GF/1978,Norovirus Hu/GI.5/E57/UG/1975, NorovirusHu/GII.4/Randwick/NSW882J/2011/AU, NorovirusHu/GII.4/Berowra/NSW767L/2012/AU, NorovirusHu/GII.4/Sydney/NSW0514/2012/AU, Norovirus Hu/GII.4/HongKong/CUHK3830/2012/CHN, Norovirus Hu/GII.4/VP1172/Shanghai/2012/CHN,Norovirus Nu/GII-4/New Taipei/CGMH61/2012/TW, NorovirusGII/Hu/HKG/2013/GII.4/CUHK-NS-141, NorovirusGII/Hu/JP/2002/GII.P12_GII.13/Saitama/TBD, NorovirusGII/Hu/JP/2001/GII.P12_GII.12/Saitama/T15, NorovirusGII/Hu/JP/2007/GII.P21_GII.21/, Kawasaki/Y0284, NorovirusGII/Hu/JP/2007/GII.P15_GII.15/Sapporo/HK299, NorovirusGI/Hu/JP/2007/GI.P3_GI.3/Shimizu/KK2866, NorovirusGII/Hu/JP/2007/GII.P7_GII.14/Fukuoka/KK282, NorovirusGI/Hu/JP/2007/GI.P8_GI.8/Nagoya/KY531, NorovirusHu/GII.4/SJTUH1/CHN/2014, Norovirus Hu/GII.4/variant Sydney 2012/FRA,Norovirus Hu/GII-4/Hokkaido4/2006/JP, NorovirusGIV/Hu/Jp/2001/GIV.1/OCO1017023, NorovirusHu/GII.4/Beijing/53671/2007/CHN, Norovirus Hu/II.4/2200661/HK/2010,Norovirus Hu/GII.4/Aichi368-14/2014, Norovirus Hu/GII.4/Hunter284E/040/AU, Norovirus Hu/GII-4/Osaka/1998/JPN, NorovirusHu/GI.1/P774.Delsjo2004/Gothenburg/Sweden, Noroviruspig/GII.11/F18-10/2005/CAN, Norovirus Hu/GII.4/Wellington/1995/USA,Norovirus Hu/GII.4/Henry/2000/USA, Norovirus Hu/GII.4/SSCS/2005/USA,Norovirus GII/Hu/IN/2006/GII.P4_GII.4_(—) Yerseke2006a/Pune-PC21,Norovirus Hu/GI.1/P7-587/2007/Stromstad/Sweden, NorovirusHu/GI.2/Leuven/2003/BEL, Norovirus Hu/GII.7/NSW743L/2008/AUS, NorovirusHu/GII.2/NF2002/USA/2002, Norovirus Hu/GII.4/NF2003/USA/2003, NorovirusHu/GII.3/1999, Norovirus Hu/GIV.1/Ahrenshoop246/DEU/2012, NorovirusHu/GII.4/Xi'an/P19/2010/CHN, Norovirus Hu/GII.4/PA363/2011/ITA,Norovirus Hu/GII.4/P3/2012/Gothenburg/Sweden, NorovirusHu/GII.4/Tanger/TM687/2011/MAR, Norovirus 12-X-2/2012/GII.P22/GII.5,Norovirus Hu/GII.4/Kobe034/2006/JP, Norovirus Hu/GGII.4/Tie1001/1995/NL,Norovirus Hu/GGII.4/DenHaag015/2000/NL, NorovirusHu/GGII.4/Schiedam018/2001/NL, Norovirus Hu/GGII.4/Apeldoorn023/2003/NL,Norovirus Hu/GGII.4/Middelburg007/2004/NL, NorovirusHu/GII-4/Matsudo/021071/2002/JP, NorovirusHu/GII-4/Kaiso/030556/2003/JP, Norovirus Hu/GII-4/Awa/040354/2004/JP,Norovirus Hu/GII.4/Apeldoorn317/2007/NL, NorovirusHu/GII.2/Rotterdam39E/2002/NL, Norovirus Hu/GII.4/RotterdamP2D0/2005/NL,Norovirus Hu/GII.4/Stockholm/19865/2008/SE, NorovirusHu/GII.6/OC04062VLP/2004/JP, Norovirus Hu/GII.4/HS194/2009/US, NorovirusHu/GII.12/HS210/2010/USA, Norovirus Hu/GI.0/8FIIa/1968/USA, NorovirusHu/GII.4/CHDC5191/1974/USA, Norovirus Hu/GII.4/N76/2010/HuZhou,Norovirus Hu/GII.6/S9c/1976/SEN, Norovirus Hu/GII.4/KL45/1978/MYS,Norovirus Hu/GII.4/NIHIC17.5/2012/USA, NorovirusHu/GII.4/NIHIC9/2011/USA, Norovirus Hu/GII.4/C110/1978/GUF, NorovirusHu/GII.4/HS66/2001/USA, NorovirusHu/GII/JP/2015/GII.P17_GII.17/Kawasaki308, NorovirusHu/GII/JP/2014/GII.P17_GII.17/Nagano8-1, NorovirusHu/GII/JP/2015/GII.Pe_GII.4/Osaka/DSF78, NorovirusGI/Hu/NL/2011/GI.4/Groningen, NorovirusGII/Hu/NL/2014/GII.4/Groningen01, NorovirusHu/GII.4/Kenepuru/NZ327/2006/NZL, NorovirusHu/GII.4/Rathmines/NSW287R/2007/AUS, NorovirusHu/GII.4/Turramurra/NSW892U/2009/AUS, NorovirusHu/GII.4/Seoul/0389/2009/KDR, Norovirus Hu/GII.4/Seoul/0945/2009/KDR,Norovirus Hu/GII.12/Shelby/2009/USA, Norovirus Hu/GI.7/TCH-060/USA/2003,Norovirus Hu/GII.1/Ascension208/2010/USA, NorovirusHu/GII.13/VAI73/2010/USA, Norovirus Hu/GII.21/Salisbury150/2011/USA,Norovirus Hu/GII.4/1997/USA, Norovirus Hu/GII.4/FarmingtonHills/2004/USA, Norovirus Hu/GII.4/Minerva/2006/USA, NorovirusHu/GII.4/Ohio/71/2012/USA, Norovirus Hu/GII.4/AlbertaEI065/2011/CA,Norovirus Hu/GII.4/SG4051-09/2009/SG, NorovirusHu/GII.3/TCH-104/USA/2002, Norovirus Hu/GI.6/TCH-099/USA/2003, Norovirus06-AM-11/2006/GII.4/Yerseke/2006a, Norovirus09-BI-2/2009/GII.4/NewOrleans/2009, Norovirus Hu/GII.4/PR328/2013/ITA,Norovirus Hu/GII.P17_GII.17/PR668/2015/ITA, NorovirusHu/GII.4/AlbertaSPI/2013/CA, Norovirus Hu/GII.4/C00007892/2011/UK,Norovirus Hu/GII.6/GZ2010-L96/Guangzhou/CHN/2011, NorovirusBo/GIII.1/Aba-Z5/2002/HUN, Norovirus GI.9, NorovirusHu/GII.17/CUHK-NS-670/HKG/2015, NorovirusGII/Hu/SI/2015/GII.17/Ljubljana1662, NorovirusHu/GII.17/CUHK-NS-647/HKG/2015, NorovirusHu/GII.21/CUHK-NS-609/HKG/2015, NorovirusHu/GII.4/Melbourne6623/2016/AUS, NorovirusGII/Hu/JP/2016/GII.P16_GII.4_(—) Sydney2012/OH16002, NorovirusHu/GII/JP/2016/GII.P16_GII.4_(—) Sydney2012/Kawasaki194, Norovirus16F2149_GII.2_Guangdong_CHN_2016, NorovirusHu/GII.17/CUHK-NS-864/HKG/2016, NorovirusGII/Hu/ZAF/2012/GII.P4_GII.4/CapeTown/9772, Norovirus GII.12, SnowMountain virus, Human calicivirus strain Melksham.

In one embodiment, the artificial nucleic acid is derived from aNorovirus selected from the group consisting of genogroup I Norovirus,genogroup II Norovirus, genogroup III Norovirus, genogroup IV Norovirus,and genogroup V Norovirus; preferably the artificial nucleic acid isderived from a Norovirus selected from the group consisting of a GI.1 toGI.17 Norovirus, GII.1 to GII.24 Norovirus, GIII.1 to GIII.4 Norovirus,GIV.1 to GIV.4 Norovirus and GV.1 to GV.4 Norovirus; more preferably,the artificial nucleic acid is derived from a Norovirus selected fromthe group consisting of GI.1 Norovirus and 011.4 Norovirus, even morepreferably, the artificial nucleic acid is derived from a GII.4Norovirus, still more preferably, the artificial nucleic acid is derivedfrom a GII.4 CIN-1 Norovirus or a GII.4 Sydney Norovirus or a GII.4Sydney 2012 Norovirus.

In preferred embodiments, the artificial nucleic acid is derived from aNorovirus selected from the group consisting of NorovirusGII.4-031093-USA-2003, Norovirus GII.4/Farmington Hills/HU/USA,Norovirus GII.4-2006b 092895-USA-2008, NorovirusGII.4-GZ2010-L87-Guangzhou-2011, Norovirus GII.4-USA-1997, NorovirusGI.1-USA-1968-Capsidprotein.

Norovirus Peptides or Proteins:

The at least one polypeptide encoded by the at least one coding regionof the inventive artificial nucleic acid comprises at least oneNorovirus protein. The RNA genome of Norovirus typically encodes aplurality of structural and non-structural proteins.

Translation of Norovirus RNA typically leads to a precursor proteincomprising a plurality of individual viral (structural andnon-structural) proteins (or precursor of these proteins) in onepolypeptide chain, which is typically referred to as “polyprotein” or“precursor protein”.

In the context of the present invention, a Norovirus polyproteintypically comprises amino acid sequences that are target sites forenzymes that specifically cleave the polyprotein in order to yieldfragments of the polyprotein, wherein the fragments preferably comprisean individual Norovirus protein or two or more Norovirus proteins, or afragment or variant thereof. In the context of the present invention,the term “polyprotein” may also refer to a polypeptide chain comprisingthe amino acid sequences of at least two individual Norovirus proteins,or a fragment or variant thereof. Cleavage of a Norovirus polyproteinpreferably occurs between individual Norovirus proteins (e.g. betweenthe capsid protein VP1 and the capsid protein VP2, or fragments orvariants thereof. An individual Norovirus protein, or a fragment orvariant thereof, e.g. as obtained from a polyprotein by cleavage, ispreferably referred to as “mature Norovirus protein”. In the context ofthe present invention, the term “mature Norovirus protein” is notlimited to an individual Norovirus protein, or a fragment or variantthereof, which was generated by cleavage of a polyprotein, but alsocomprises an individual Norovirus protein of another origin, such as anindividual Norovirus protein expressed recombinantly from an artificialnucleic acid. Preferably, a mature Norovirus protein lacks an amino acidsequence that is typically present in a corresponding amino acidsequence encoding said Norovirus protein in a Norovirus polyprotein(precursor protein) and wherein said amino acid sequence lacking in themature Norovirus protein preferably corresponds to an amino acidsequence, which is usually removed by cleavage during processing of aNorovirus polyprotein. For example, an amino acid sequence, which is atarget site for a protease, may be present in a Norovirus polyprotein,but may be absent from a mature Norovirus protein derived from saidNorovirus polyprotein.

In one embodiment, the artificial nucleic acid comprising at least onecoding region encoding at least one polypeptide comprises at least oneNorovirus capsid protein VP1 or Norovirus capsid protein VP2 and/or afragment or a variant thereof.

In another embodiment, the artificial nucleic acid comprising at leastone coding region encoding at least one polypeptide comprises at leastone Norovirus capsid protein VP1 and/or a fragment or variant thereof.In a further embodiment, the artificial nucleic acid comprises aNorovirus capsid protein VP1 and/or a fragment or variant thereof.

In the context of the present invention, the term “Norovirus protein”may refer to any amino acid encoded by a Norovirus nucleic acid. In thecontext of the invention, a Norovirus capsid protein VP1 or VP1 proteinis preferred. For example, a “Norovirus protein” may be any polypeptidecomprising or consisting of an amino acid sequence according to any oneof the following amino acid sequences from Genbank, or a fragment orvariant of any of these sequences as provided in Table 1 (Column 2;“NCBI or Genbank Accession No.”) and Table 3 (Column 2; “NCBI or GenbankAccession No.”).

In particular, the term “Norovirus protein” as used herein comprises anindividual structural or non-structural Norovirus protein. For example,a Norovirus protein in the meaning of the present invention may be aprotein selected from the group consisting of Norovirus capsid proteinVP1, Norovirus capsid protein VP2, and a Norovirus non-structuralprotein (NS), such as NS1/NS2, NS3, NS4, NS5, NS6, or NS7.

In particular, the term “Norovirus protein” as used herein is aNorovirus capsid protein VP1. Further, in particular, the term“Norovirus protein” as used herein is a Norovirus capsid protein derivedfrom a GII.4 Norovirus.

As used herein, the term “Norovirus protein” may also refer to an aminoacid sequence corresponding to an individual Norovirus protein aspresent in a Norovirus polyprotein (precursor protein). Said amino acidsequence in the polyprotein may differ from the amino acid sequence ofthe corresponding amino acid sequence of the respective mature Norovirusprotein (i.e. after cleavage/processing the polyprotein). For example,the corresponding amino acid sequence comprised in the polyprotein maycomprise amino acid residues that are removed during cleavage/processingof the polyprotein (such as a signal sequence or a target site for aprotease) and that are no longer present in the respective matureNorovirus protein. In the context of the present invention, the term“Norovirus protein” comprises both, the precursor amino acid sequencecomprised in a Norovirus polyprotein (i.e. as part of a polypeptidechain optionally further comprising other viral proteins) as well as therespective mature individual Norovirus protein. For example, the term“Norovirus capsid protein VP1” as used herein may refer to an amino acidsequence in a Norovirus polyprotein corresponding to the precursorsequence of Norovirus capsid protein VP1 (comprising, for example, a(C-terminal) signal sequence) as present in a Norovirus polyprotein aswell as to a mature (separate) Norovirus capsid protein VP1 (no longercomprising, for example, a (C-terminal) signal sequence).

Where reference is made to amino acid residues and their position in aNorovirus protein or in a Norovirus polyprotein, any numbering usedherein—unless stated otherwise—relates to the position of the respectiveamino acid residue in a Norovirus polyprotein (precursor protein),wherein position “1” corresponds to the first amino acid residue, i.e.the amino acid residue at the N-terminus of a Norovirus polyprotein.More preferably, the numbering with regard to amino acid residues refersto the respective position of an amino acid residue in a Noroviruspolyprotein, which is preferably derived from a Norovirus strainselected from the group consisting of Genogroup II or Genogroup I(abbreviated as GII, or GI, respectively), more preferably from thegroup consisting of Genogroup II genotype 4 (abbreviated as GII.4) orGenogroup I genotype 1 (abbreviated as GII.4 and GI.1), or even morepreferably the strain is selected from strain Norovirus strain GII.4CIN-1 or CIN-002.

Preferred Norovirus protein in the context of the invention may be anypolypeptide comprising or consisting of an amino acid sequence accordingto any one of the following amino acid sequences from Genbank, or afragment or variant of any of these sequences: ACO55068, AFS33552,AFS33555, AFX71665, BAI49904, BAI49914, BAS02083, CRL46958, CRL46973,ADB27027, AGI96397, AGX01095, AGX01098, AKI30060, AGL98413, ACT76145,ACT76148, ACT76151, AED02034, AEX10549, AFJ21448, AFN06726, AFN06727,AFN06731, AFN06732, AFN06733, AFN06735, AFV08771, AFV08777, AFV08795,AFX95940, AFJ99552, AGK25912, AID68581, AII73717, AII73735, AII73741,AII73759, AII73765, AII73780, AII73783, AIS40019, AIY27747, BAG70437,BAU16306 ACY00615, ADK47170, BAQ20801, AAZ31376, ABI97981, ABW74128,ACC69023, ACL27297, ACL27298, ACL27299, ACL31322, ACN32770, ACU156258,ACX85810, AFB18010, AFB18013, AFK75854, AFN61315, AFQ00511, AHC12655,AHZ12912, AIC32559, AID51489, BAF45861, BAF74508, BAF74509, BAF74512,BAF74517, BAF74521, BAF95499, BAF95501, BAF95505, BAG55289, BAG68713,BAG68801, BAH30707, BAL40873, ADB27914, ADT70684, AFJ38516, AFJ38519,AFW15943, AGE99599, AGE99812, AGT17839, AFX71669, AGE99607, AH159166,BAR42290, BAR63722, BAS02084, CRL46953, CRL46962, ABQ63283, ACW19927,ADQ43783, ADV37805, ADV37919, AEI83469, AEQ77282, AFA55174, AFC89656,AFC89665, AFJ04707, AFJ04708, AFJ04709, AFP89593, AFU55731, AFU92710,AGO64038, AGT62521, AJZ77004, AJZ77015, AKE31861, ALD09618, ALT54494,CCX28619, AGC96535, ABY67257, AHA91656, KT315718, KT591501, KT315706,KR921940, KX767083, LC153121, LC175468, KY485125, KU555841, KP784696,KP064099, U70059, X81879.

Preferred examples of Noroviruses which may be used for providing thenucleic acid molecules of the invention may include NorovirusGII.4-031693-USA-2003, Norovirus GII.4/Farmington Hills/2002/USA,Norovirus GII.4-2006b 092895-USA-2008, NorovirusGII.4-GZ2010-L87-Guangzhou-2011, Norovirus GII.4-USA-1997, NorovirusGI.1-USA-1968.

Further Preferred examples of Noroviruses which may be used forproviding the nucleic acid molecules of the invention may includeNorovirus Hu/GII.4/Dijon/E872/2002/FRA, NorovirusHu/GII.4/MD120-12/1987/USA, Norovirus Hu/GII.1/7EK/Hawaii/1971/USA,Norovirus Hu/GII.6/CHDC4073/1984/USA, NorovirusHu/GII.4/Hiroshima/19/2001/JPN, NorovirusHu/GII.4/Hiroshima/67/2006/JPN, NorovirusHu/GII/JP/2015/GII.PeGII.4/Osaka/OSF78, NorovirusGII/Hu/NL/2012/GII.4/Groningen, NorovirusGII/Hu/NL/2014/GII.2/Groningen, Norovirus Hu/GII.4/NewOrleans1500/2008/USA, Norovirus Hu/GII.6/Ohio/490/2012/USA, NorovirusHu/GII.3/Jingzhou/2013402/CHN, Norovirus Hu/GII.4/Jingzhou/2013403/CHN,Norovirus Hu/GII.17/Gaithersburg/2014/113, NorovirusHu/GII.4/C127/GF/1978, Norovirus Hu/GII.4/CHDC3967/1988/US, NorovirusHu/GII.4/CHDC4108/1987/US, Norovirus Hu/GII.4/CHDC4871/1977/113,Norovirus Hu/GII.3/CHDC5261/1990/US, NorovirusHu/GII.3/Milwaukee009/2010/USA, NorovirusHu/GII.4/Miranda/NSW817L/2010/AU, Norovirus Hu/GII.2/KL109/MY/1978,Norovirus Hu/GII.14/HK74/CN/1978, Norovirus Hu/GII.7/HK4/CN/1976,Norovirus Hu/GII.17/C142/GF/1978, Norovirus Hu/GII.5/C15/GF/1978,Norovirus Hu/GI.5/E57/110/1975, NorovirusHu/GII.4/Randwick/NSW882J/2011/AU, NorovirusHu/GII.4/Berowra/NSW767L/2012/AU, NorovirusHu/GII.4/Sydney/NSW0514/2012/AU, Norovirus Hu/GII.4/HongKang/CUHK3630/2012/CHN, Norovirus Hu/GII.4/VP1172/Shanghai/2012/CHN,Norovirus Hu/GII-4/New Taipei/CGMH61/2012/TW, NorovirusGII/Hu/HKG/2013/GII.4/CUHK-NS-141, NorovirusGII/Hu/JP/2002/GII.P12_GII.13/Saitama/T80, NorovirusGII/Hu/JP/2001/GII.P12_GII.12/Saitama/T15, NorovirusGII/Hu/JP/2007/GII.P21_GII.21/, Kawasaki/YO284, NorovirusGII/Hu/JP/2007/GII.P15_GII.15/Sapporo/HK299, NorovirusGI/Hu/JP/2007/GI.P3_GI.3/Shimizu/KK2866, NorovirusGII/Hu/JP/2007/GII.P7_GII.14/Fukuoka/KK282, NorovirusGI/Hu/JP/2007/GI.P8_GI.8/Nagoya/KY531, NorovirusHu/GII.4/SJTLIH1/CHN/2014, Norovirus Hu/GII.4/variant Sydney 2012/FRA,Norovirus Hu/GII-4/Hokkaido4/2006/JP, NorovirusGIV/Hu/Jp/2001/GIV.1/OC01017023, NorovirusHu/GII.4/Beijing/53671/2007/CHN, Norovirus Hu/II.4/2200661/HK/2010,Norovirus Hu/GII.4/Aichi368-14/2014, Norovirus Hu/GII.4/Hunter284E/040/All, Norovirus Hu/GII-4/Osaka/1998/JPN, NorovirusHu/GI.1/P774.Delsjo2004/Gothenburg/Sweden, Noroviruspig/GII.11/F18-10/2005/CAN, Norovirus Hu/GII.4/Wellington/1995/USA,Norovirus Hu/GII.4/Henry/2000/USA, Norovirus Hu/GII.4/SSCS/2005/USA,Norovirus GII/Hu/IN/2006/GII.P4_GII.4_(—) Yerseke2006a/Pune-PC21,Norovirus Hu/GI.1/P7-587/2007/Stromstad/Sweden, NarovirusHu/GI.2/Leuven/2003/BEL, Norovirus Hu/GII.7/NSW743L/2008/AUS, NorovirusHu/GII.2/NF2002/USA/2002, Norovirus Hu/GII.4/NF2003/USA/2003, NorovirusHu/GII.3/1999, Norovirus Hu/GIV.1/Ahrenshoop246/DEU/2012, NorovirusHu/GII.4/Xi'an/P19/2010/CHN, Norovirus Hu/GII.4/PA363/2011/ITA,Norovirus Hu/GII.4/P3/2012/Gothenburg/Sweden, NorovirusHu/GII.4/Tanger/TM987/2011/MAR, Norovirus 12-X-2/2012/GII.P22/GII.5,Norovirus Hu/GII.4/Kobe034/2009/JP, Norovirus Hu/GGII.4/Tiel001/1995/NL,Norovirus Hu/GGII.4/DenHaag015/2000/NL, NorovirusHu/GGII.4/Schiedam018/2001/NL, Norovirus Hu/GGII.4/Apeldoorn023/2003/NL,Norovirus Hu/GGII.4/Middelburg007/2004/NL, NorovirusHu/GII-4/Matsuda/021071/2002/JP, NorovirusHu/GII-4/Kaiso/030559/2003/JP, Norovirus Hu/GII-4/Awa/040354/2004/JP,Norovirus Hu/GII.4/Apeldoorn317/2007/NL, NorovirusHu/GII.2/Rotterdam39E/2002/NL, Norovirus Hu/GII.4/RotterdamP200/2005/NL,Norovirus Hu/GII.4/Stockholm/19865/2008/SE, NorovirusHu/GII.6/OC04092VLP/2004/JP, Norovirus Hu/GII.4/HS194/2009/US, NorovirusHu/GII.12/HS210/2010/USA, Norovirus Hu/GI.1/8FIIa/1998/USA, NorovirusHu/GII.4/CHDC5191/1974/USA, Norovirus Hu/GII.4/N76/2010/HuZhou,Norovirus Hu/GII.9/39c/1976/SEN, Norovirus Hu/GII.4/KL45/1978/MYS,Norovirus Hu/GII.4/NIHIC17.5/2012/USA, NorovirusHu/GII.4/NIHIC9/2011/USA, Norovirus Hu/GII.4/C110/1978/GUF, NorovirusHu/GII.4/HS66/2001/USA, NorovirusHu/GII/JP/2015/GII.P17_GII.17/Kawasaki308, NorovirusHu/GII/JP/2014/GII.P17_GII.17/Nagano8-1, NorovirusHu/GII/JP/2015/GII.Pe_GII.4/Dsaka/OSF78, NorovirusGI/Hu/NL/2011/GI.4/Groningen, NorovirusGII/Hu/NL/2014/GII.4/Groningen01, NorovirusHu/GII.4/Kenepuru/NZ327/2006/NZL, NorovirusHu/GII.4/Rathmines/NSW287R/2007/AUS, NorovirusHu/GII.4/Turramurra/NSW892U/2009/AUS, NorovirusHu/GII.4/Seoul/0389/2009/KOR, Norovirus Hu/GII.4/Seoul/0945/2009/KOR,Norovirus Hu/GII.12/Shelby/2009/USA, Norovirus Hu/GI.7/TCH-060/USA/2003,Norovirus Hu/GII.1/Ascension208/2010/USA, NorovirusHu/GII.13/VA173/2010/USA, Norovirus Hu/GII.21/Salisbury150/2011/USA,Norovirus Hu/GII.4/1997/USA, Norovirus Hu/GII.4/FarmingtonHills/2004/USA, Norovirus Hu/GII.4/Minerva/2009/USA, NorovirusHu/GII.4/Ohio/71/2012/USA, Norovirus Hu/GII.4/AlbertaEl095/2011/CA,Norovirus Hu/GII.4/SG4051-09/2009/SG, NorovirusHu/GII.3/TCH-104/USA/2002, Norovirus Hu/GINTCH-099/USA/2003, Norovirus06-AM-11/2009/GII.4/Yerseke/2006a, Norovirus09-BI-2/2009/GII.4/NewOrleans/2009, Norovirus Hu/GII.4/PR328/2013/ITA,Norovirus Hu/GII.P17_GII.17/PR668/2015/ITA, NorovirusHu/GII.4/AlbertaSPI/2013/CA, Norovirus Hu/GII.4/C00007892/2011/UK,Norovirus Hu/GII.6/GZ2010-L96/Guangzhou/CHN/2011, NorovirusBo/GIII.1/Aba-Z5/2002/HUN, Norovirus GI.9, NorovirusHu/GII.17/CUHK-NS-670/HKG/2015, NorovirusGII/Hu/SI/2015/GII.17/Ljubljana1662, NorovirusHu/GII.17/CUHK-NS-947/HKG/2015, NorovirusHu/GII.21/CUHK-NS-909/HKG/2015, NorovirusHu/GII.4/Melbourne6623/2016/AUS, NorovirusGII/Hu/JP/2016/GII.P16_GII.4_(—) Sydney2012/OH16002, NorovirusHu/GII/JP/2016/GII.P16_GII.4_(—) Sydney2012/Kawasaki194, Norovirus16F2149_GII.2_Guangdong_CHN_2016, NorovirusHu/GII.17/CHK-NS-864/HKG/2016, NorovirusGII/Hu/ZAF/2012/GII.P4_GII.4/CapeTown/9772, Norovirus GII.12, SnowMountain virus, Human calicivirus strain Melksham.

Preferred examples of Noroviruses which may be used for providing thenucleic acid molecules of the invention are provided in Table 1 (Column6 “Strain/Isolate”). Even more preferred examples of Noroviruses whichmay be used for providing the nucleic acid molecules of the inventionare provided in Table 3 (Column 1“Strain/Isolate”).

The nucleic acid and corresponding amino acid sequences of each are allincorporated by reference in their entirety. In same embodiments, acryptogram can be used for identification purposes and is organized:host species from which the virus was isolated/genusabbreviation/species abbreviation/strain name/year of occurrence/countryof origin. (Green et al., Human Caliciviruses, in Fields Virology Vol. 1841-874 (Knipe and Howley, editors-in-chief, 4th ed., LippincottWilliams 6. Wilkins 2000). Use of a combination of Norovirus genogroupssuch as a genogroup I.1 (Norwalk virus) and II.4 (f.e. Houston virus) orother commonly circulating strains, or synthetic constructs representingcombinations or portions thereof are preferred in some embodiments. Newstrains of Noroviruses are routinely identified (Centers for DiseaseControl, Morbidity and Mortality Weekly Report, 56(33):842-846 (2007))and consensus sequences of two or more viral strains may also be used toexpress Norovirus antigens.

In some embodiments described herein, the at least one polypeptideencoded by the at least one coding region of the artificial nucleic acidmay consist of an individual Norovirus protein, the amino acid sequenceof which does typically not comprise an N-terminal methionine residue.It is thus understood that the phrase “polypeptide consisting ofNorovirus protein . . . ” relates to a polypeptide comprising the aminoacid sequence of said Norovirus protein and—if the amino acid sequenceof the respective Norovirus protein does not comprise such an N-terminalmethionine residue—an N-terminal methionine residue.

Norovirus Sequences:

According to a preferred embodiment, the inventive artificial nucleicacid comprises at least one coding region encoding at least onepolypeptide comprising or consisting of at least one Norovirus proteinas described herein, wherein the at least one Norovirus proteincomprises an amino acid sequence according to any one of SEQ ID NOs:1-4410, or a fragment or variant of any of these sequences.

In one embodiment, the term “sequence”, “sequence of the invention”,“artificial nucleic acid” or “artificial nucleic acid of the invention”refers to any one of SEQ ID NOs: 1-39590, 39713-39746.

In a further embodiment, the artificial nucleic acid of the inventioncomprises at least one encoded polypeptide comprising

-   -   (i) at least one of the amino acid sequences according to any        one of SEEM NOs: 1-4410; and/or    -   (ii) at least one of the amino acid sequences having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        amino acid sequence represented by any one of SKI ID NOs:        1-4410; and/or    -   (iii) an orthologue or a paralogue of any one of SEQ ID NOs:        1-39690, 39713-39746; and/or a fragment or variant of any of        these sequences.

In another embodiment the artificial nucleic acid of the inventioncomprises at least one coding region comprising

-   -   (i) at least one of the nucleic acid sequences according to any        one of SEQ ID NOs: 4411-39990, 39713-39749; and/or    -   (ii) at least one of the nucleic acid sequences having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 91%, 62%, 63%, 64%, 95%, 66%,        97%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        nucleic acid sequence represented by any one of SEQ ID NOs:        4411-39890, 39713-39748; and/or    -   (iii) at least one complement of the nucleic acid sequences        which are capable of hybridizing with a nucleic acid sequence        comprising a sequence as shown in SEQ ID NOs: 4411-39690,        39713-39748, and/or to a nucleic acid encoding a polypeptide        having a sequence as shown in SEQ ID NOs: 1-4410, and/or    -   (iv) an orthologue or a paralogue of any one of SEQ ID NOs:        1-39890, 39713-39746; and/or a fragment or variant of any of        these sequences.

In the context of the present invention a fragment of a protein or avariant thereof encoded by the at least one coding sequence of theartificial nucleic acid according to the invention may typicallycomprise an amino acid sequence having a sequence identity of at least5%, 10%, 20%, 30%, 40%, 50%, 80%, 70%, 80%, 85%, 88%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 99%, 97%, 98%, or 99%, preferably of atleast 70%, more preferably of at least 80%, even more preferably atleast 85%, even more preferably of at least 90% and most preferably ofat least 95% or even 97%, with an amino acid sequence of the respectivenaturally occurring full-length protein or a variant thereof, preferablyas disclosed in Table 1, column 1, column 2 or column 3, more preferablyas disclosed in Table 3, column 1, column 2, column 3.

In a preferred embodiment, the at least one coding sequence of theartificial nucleic acid sequence according to the invention preferablyencodes Norovirus proteins selected from the proteins provided in Table1, or a fragment or variant thereof. Any Norovirus protein provided inTable 1, or any a fragment or variant thereof, can cause an immuneresponse when administered to an individual. Therefore, all Norovirusproteins provided in Table 1 and Table 3 can be considered as preferredNorovirus antigens in the context of the present invention.

It is further preferred that the at least one coding sequence of theartificial nucleic acid sequence of the present invention encodes aNorovirus protein or peptide, or a fragment or variant thereof, whereinthe Norovirus protein or peptide is an antigen selected from theantigens listed in Table 1. Therein, each row (row 1-row 4410)corresponds to a Norovirus protein or antigen as identified by therespective gene name (first column, column 1 “Name”) and the databaseaccession number of the corresponding protein (second column, column 2“NCBI or Genbank Accession No.”). The third column, column 3 (“A”) inTable 1 indicates the SEQ ID NOs corresponding to the respective aminoacid sequence as provided herein. The SEQ ID Hs corresponding to thenucleic acid sequence of the wild type nucleic acid sequence encodingthe Norovirus protein or peptide is indicated in the fourth column,column 4 (“B”). The fifth column, column 5 (“C”) provides the SEQ ID NOscorresponding to modified nucleic acid sequences of the nucleic acidsequences as described herein that encode the Norovirus protein orpeptide preferably having the amino acid sequence as defined by the SEQID NOs indicated in the third column (“A”) or by the database entryindicated in the second column (“NCBI or Genbank Accession No.”).

In this context it is further preferred that the at least one codingsequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Norovirus protein or peptide which isderived from Norovirus polyprotein, or a fragment or variant thereof,wherein the Norovirus polyprotein is selected from the Noroviruspolyprotein amino acid sequences listed in Table 1. Therein, each rowcorresponds to a Norovirus polyprotein as identified by the respectivegene name (first column “Name”, derived from NCBI or Genbank) and thedatabase accession number of the corresponding protein (second column“NCBI or Genbank Accession No.”). The third column (“A”) in Table 1indicates the SEQ ID NOs corresponding to the respective amino acidsequence as provided herein. The SEQ ID NOs corresponding to the nucleicacid sequence of the wild type nucleic acid encoding the Norovirusprotein or peptide is indicated in the fourth column (“B”). The fifthcolumn (“C”) provides the SEQ ID NOs corresponding to modified nucleicacid sequences of the nucleic acids as described herein that encode theNorovirus protein or peptide preferably having the amino acid sequenceas defined by the SEQ ID NOs indicated in the third column (“A”) or bythe database entry indicated in the second column (“NCBI or GenbankAccession No.”).

Particularly preferred in this context are the Norovirus polyprotein andnucleic acid sequences according to SEQ ID NOs: 39713-39746.

In specific embodiments the Norovirus protein or peptide is derived froma Norovirus capsid protein VP1 according to SEQ ID NOs: 1-4410.

In this context it is further preferred that the at least one codingsequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Norovirus protein or peptide which isderived from Norovirus capsid protein VP1, or a fragment or variantthereof, wherein the Norovirus capsid protein VP1 is selected from theNorovirus capsid protein VP1 amino acid sequences listed in Table 1.Therein, each row corresponds to a Norovirus capsid protein VP1 asidentified by the respective gene name (first column “Name”) and thedatabase accession number of the corresponding protein (second column“NCBI or Genbank Accession No”). The third column (“A”) in Table 1indicates the SEQ ID NOs corresponding to the respective amino acidsequence as provided herein. The SEQ ID NOs corresponding to the nucleicacid sequence of the wild type RNA encoding the Norovirus antigen isindicated in the fourth column (“B”). The fifth column (“C”) providesthe SEQ ID NOs corresponding to modified nucleic acid sequences of theRNAs as described herein that encode the Norovirus antigen preferablyhaving the amino acid sequence as defined by the SEQ ID NOs indicated inthe third column (“A”) or by the database entry indicated in the secondcolumn (“NCBI or Genbank Accession No”).

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Norovirus protein or peptide as described herein. Preferably,the inventive artificial nucleic acid comprises or consists of a codingsequence according to any one of SEQ ID NOs: 4411-39690, 39713-39746, ora homolog, fragment or variant of any of these sequences (see Table 1,column “B” and “C”).

Accordingly, in another embodiment, a nucleic acid molecule of theinvention is at least 15, 20, 25 or 30 nucleotides in length.Preferably, it hybridizes under stringent conditions to a nucleic acidmolecule comprising a nucleotide sequence of the nucleic acid moleculeof the present invention or used in the methods of the presentinvention. The nucleic acid molecule is preferably at least 20, 30, 50,100, 250 or more nucleotides in length.

In a preferred embodiment, the present invention thus providesartificial nucleic acid sequences comprising at least one codingsequence, wherein the coding sequence encoding Norovirus capsid proteinVP1 comprises or consists any one of the nucleic acid sequences definedin Table 1, preferably in the fourth or fifth column (column “B” or “C”,respectively) of Table 1, or a fragment or variant of any one of thesesequences.

In particularly preferred embodiments the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Noroviruscapsid protein VP1 according to SEQ ID NOs: 4411-39690, 39713-39746.

In these context it is particularly preferred that the nucleic acidsequence according to the invention comprises at least one codingsequence encoding Norovirus capsid protein VP1 comprising a nucleic acidsequence selected from sequences being identical or at least 50%, 60%,70%, 80%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequences as disclosed inTable 1, preferably in the fourth or fifth column (column “B” or “C”,respectively) of Table 1 or a fragment or variant thereof.

According to a particularly preferred embodiment, the present inventionprovides an nucleic acid sequence as defined herein comprising at leastone coding sequence encoding at least one Norovirus peptide or proteinderived from Norovirus capsid protein VP1, wherein the coding sequencecomprises or consists of any one of the (modified) nucleic acidsequences defined in the Column “C” of Table 1, or of a fragment orvariant of any one of these sequences.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Norovirus protein or peptide as described herein, wherein theat least one Norovirus protein comprises an amino acid sequenceaccording to any one of SEQ ID NOs: 1-4410, or a homolog, fragment orvariant of any of these sequences (see Table 1, third column, column“A”). According to a preferred embodiment, the inventive artificialnucleic acid comprises at least one coding sequence encoding at leastone protein or peptide derived from a Norovirus, or a fragment orvariant thereof, wherein the Norovirus protein or peptide preferablycomprises or consists of any one of the amino acid sequences defined inthe third column (column “A”) of Table 1, or a fragment or variant ofany one of these sequences. In other words, the at least one codingsequence preferably encodes a Norovirus protein comprising or consistingof an amino acid sequence selected from the group consisting of SEQ IDNOs: 1-4410.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence according to the invention comprises orconsists of an nucleic acid sequence identical to or having a sequenceidentity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,preferably of at least 70%, more preferably of at least 80%, even morepreferably at least 85%, even more preferably of at least 90% and mostpreferably of at least 95% or even 97%, with any one of the (G/Cmodified) RNA sequences defined in the fifth column (column “C”) ofTable 1, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence according to the invention comprises orconsists of an nucleic acid sequence identical to or having a sequenceidentity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,preferably of at least 70%, more preferably of at least 80%, even morepreferably at least 85%, even more preferably of at least 90% and mostpreferably of at least 95% or even 97%, with any one of the (human codonusage adapted) RNA sequences defined in the fifth column (column “C”) ofTable 1, or of a fragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence according to the invention comprisesor consists of an nucleic acid sequence having a sequence identity of atleast 80% with any one of the (human codon usage adapted) RNA sequencesdefined in the fifth column (column “C”) of Tables 1, or of a fragmentor variant of any one of these sequences.

According to a particularly preferred embodiment, the present inventionprovides an nucleic acid sequence as defined herein comprising at leastone coding sequence encoding at least one Norovirus peptide or proteinderived from Norovirus capsid protein VP1, wherein the coding sequencecomprises or consists of any one of the (human codon usage adapted) RNAsequences defined in the fifth column (column “C”) of Table 1, or of afragment or variant of any one of these sequences.

Norovirus peptide or protein derived from Norovirus capsid protein VP1,wherein the coding sequence comprises or consists of any one of the(codon optimized) RNA sequences defined in the fifth column (column “C”)of Table 1, or of a fragment or variant of any one of these sequences.

Accordingly, in another embodiment, a nucleic acid molecule of theinvention is at least 15, 20, 25 or 30 nucleotides in length.Preferably, it hybridizes under stringent conditions to a nucleic acidmolecule comprising a nucleotide sequence of the nucleic acid moleculeof the present invention or used in the methods of the presentinvention, e.g. comprising the sequence shown in SEQ ID NOs: 4411-39690,39713-39746. The nucleic acid molecule is preferably at least 20, 30,50, 100, 250 or more nucleotides in length.

In one embodiment of the invention, a complement sequence derived fromSEQ ID NOs: 4411-39690, 39713-39746, or a fragment or variant thereof isused in the hybridization. Preferably, a complement of a nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence shown in SEQ ID NOs: 4411-39690, 39713-39749 correspond to anaturally-occurring nucleic acid molecule of the invention. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence according to the invention comprises orconsists of an nucleic acid sequence identical to or having a sequenceidentity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,preferably of at least 70%, more preferably of at least 80%, even morepreferably at least 85%, even more preferably of at least 90% and mostpreferably of at least 95% or even 97%, with any one of the (codonoptimized) RNA sequences defined in the fifth column 5 (column “C”) ofTable 1, or of a fragment or variant of any one of these sequences.

In the context of the present invention, a “fragment” of an amino acidsequence, such as a polypeptide or a protein, e.g. the at least oneNorovirus protein as described herein, may typically comprise a sequenceof a protein or peptide as defined herein, which is, with regard to itsamino acid sequence (or the respective coding nucleic acid molecule),N-terminally and/or C-terminally truncated compared to the amino acidsequence of the original (native) protein (or respective coding nucleicacid molecule). Such truncation may thus occur either on the amino acidlevel or correspondingly on the nucleic acid level. A sequence identitywith respect to such a fragment as defined herein may thereforepreferably refer to the entire protein or peptide as defined herein orto the entire (coding) nucleic acid molecule of such a protein orpeptide.

Preferably, a fragment of an amino acid sequence comprises or consistsof a continuous stretch of amino acid residues corresponding to acontinuous stretch of amino acid residues in the protein the fragment isderived from, which represents at least 5%, 10%, 20%, preferably atleast 30%, more preferably at least 40%, more preferably at least 50%,even more preferably at least 60%, even more preferably at least 70%,and most preferably at least 80% of the total (i.e. full-length)protein, from which the fragment is derived.

In the context of the present invention, a fragment of a protein or of apeptide may furthermore comprise a sequence of a protein or peptide asdefined herein, which has a length of for example at least 5 aminoacids, preferably a length of at least 6 amino acids, preferably atleast 7 amino acids, more preferably at least 8 amino acids, even morepreferably at least 9 amino acids; even more preferably at least IDamino acids; even more preferably at least II amino acids; even morepreferably at least 12 amino acids; even more preferably at least 13amino acids; even more preferably at least 14 amino acids; even morepreferably at least 15 amino acids; even more preferably at least 16amino acids; even more preferably at least 17 amino acids; even morepreferably at least 18 amino acids; even more preferably at least 19amino acids; even more preferably at least 20 amino acids; even morepreferably at least 25 amino acids; even more preferably at least 30amino acids; even more preferably at least 35 amino acids; even morepreferably at least 50 amino acids; or most preferably at least 100amino acids. For example such fragment may have a length of about 6 toabout 20 or even more amino acids, e.g. fragments as processed andpresented by MHC class 1 molecules, preferably having a length of about8 to about ID amino acids, e.g. 8, 9, or ID, (or even 6, 7, 11, or 12amino acids), or fragments as processed and presented by MHC class IImolecules, preferably having a length of about 13 or more amino acids,e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even mare amino acids, whereinthese fragments may be selected from any part of the amino acidsequence. These fragments are typically recognized by T-cells in form ofa complex consisting of the peptide fragment and an MHC molecule, i.e.the fragments are typically not recognized in their native form.Fragments of proteins or peptides may comprise at least one epitope ofthose proteins or peptides. Furthermore also domains of a protein, likethe extracellular domain, the intracellular domain or the transmembranedomain and shortened or truncated versions of a protein may beunderstood to comprise a fragment of a protein.

As used herein, a “variant” of a protein or a peptide may be generated,having an amino acid sequence, which differs from the original sequencein one or more mutation(s), such as one or more substituted, insertedand/or deleted amino acid(s). Preferably, these fragments and/orvariants have the same biological function or specific activity comparedto the full-length native protein, e.g. its specific antigenic property.“Variants” of proteins or peptides as defined in the context of thepresent invention may comprise conservative amino acid substitution(s)compared to their native, i.e. non-mutated physiological, sequence.Those amino acid sequences as well as their encoding nucleotidesequences in particular fall under the term variants as defined herein.Substitutions in which amino acids, which originate from the same class,are exchanged for one another are called conservative substitutions. Inparticular, these are amino acids having aliphatic side chains,positively or negatively charged side chains, aromatic groups in theside chains or amino acids, the side chains of which can enter intohydrogen bridges, e.g. side chains which have a hydroxyl function. Thismeans that e.g. an amino acid having a polar side chain is replaced byanother amino acid having a likewise polar side chain, or, for example,an amino acid characterized by a hydrophobic side chain is substitutedby another amino acid having a likewise hydrophobic side chain (e.g.serine (threonine) by threonine (serine) or leucine (isoleucine) byisoleucine (leucine)). Insertions and substitutions are possible, inparticular, at those sequence positions which cause no modification tothe three-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

In the context of the present invention, a “variant” of a protein orpeptide may have at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 92%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% aminoacid identity over a stretch of at least 10, at least 20, at least 30,at least 50, at least 75 or at least 100 amino acids of such protein orpeptide. More preferably, a “variant” of a protein or peptide as usedherein is at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, even more preferably at least 90%, most preferably at least 95%identical to the protein or peptide, from which the variant is derived.

Alternatively or additionally, a protein of the invention, a capsidprotein, a Norovirus capsid protein VP1 or a Norovirus capsid proteinVP2 as defined herein refers to any polypeptide being identical orhaving in increasing order of preference at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% or more amino acid sequence identity to theprotein of the invention, a capsid protein, a Norovirus capsid proteinVP1 or a Norovirus capsid protein VP2 as represented by SEQ ID NOs:1-4410, or to any of the full length polypeptide sequences given in SEQID NOs: 1-4410.

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by a nucleic acid, may also comprise those sequences,wherein nucleotides of the encoding nucleic acid sequence are exchangedaccording to the degeneration of the genetic code, without leading to analteration of the respective amino acid sequence of the protein orpeptide, i.e. the amino acid sequence or at least part thereof may notdiffer from the original sequence in one or more mutation(s) within theabove meaning.

According to a preferred embodiment, the at least one coding region ofthe inventive artificial nucleic acid comprises or consists of at leastone nucleic acid sequence according to any one of SEQ ID NOs:4411-39690, 39713-39746, or a fragment or variant of any of thesesequences.

As used herein, a “fragment” of a nucleic acid sequence comprises orconsists of a continuous stretch of nucleotides corresponding to acontinuous stretch of nucleotides in the full-length nucleic acidsequence which is the basis for the nucleic acid sequence of thefragment, which represents at least 20%, preferably at least 30%, morepreferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, and most preferably at least 90% of thefull-length nucleic acid sequence. Such a fragment, in the sense of thepresent invention, is preferably a functional fragment of thefull-length nucleic acid sequence.

In the context of the present invention, the phrase “variant of anucleic acid sequence” typically relates to a variant of a nucleic acidsequence, which forms the basis of a nucleic acid sequence. For example,a variant nucleic acid sequence may exhibit one or more nucleotidedeletions, insertions, additions and/or substitutions compared to thenucleic acid sequence, from which the variant is derived. Preferably, avariant of a nucleic acid sequence is at least 40%, preferably at least50%, more preferably at least 60%, more preferably at least 70%, evenmore preferably at least 80%, even more preferably at least 90%, mostpreferably at least 95% identical to the nucleic acid sequence thevariant is derived from. Preferably, the variant is a functionalvariant. A “variant” of a nucleic acid sequence may have at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide identity over a stretchof at least 10, at least 20, at least 30, at least 50, at least 75 or atleast 100 nucleotides of such nucleic acid sequence.

Alternatively or additionally, a nucleic acid sequence encoding aprotein of the invention, a capsid protein, a Norovirus capsid proteinVP1 or a Norovirus capsid protein VP2 as defined herein refers to anynucleic acid sequence having in increasing order of preference at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%. 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity tothe nucleic acid sequence as represented by SEQ ID NOs: 4411-39690,39713-39746, or to any of the full length polypeptide sequences given inSEQ ID NOs: 1-4410.

Preferably, the at least one polypeptide encoded by the at least onecoding region of the inventive artificial nucleic acid comprises orconsists of Norovirus capsid protein VP1, or a fragment or variantthereof. More preferably, the at least one encoded polypeptide comprisesor consists of an amino acid sequence according to any one of SEQ IDNOs: 1-4410, or a fragment or variant of any of these sequences.

Alternatively or additionally, a nucleic acid sequence encoding aNorovirus capsid protein VP1 as defined herein refers to any nucleicacid sequence being identical or having in increasing order ofpreference at least 50%, 51%, 52%, 53%. 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99% or moresequence identity to the full-length nucleic acid sequence asrepresented by SEQ ID NOs: 4411-39690, 39713-39746.

In certain embodiments, the at least one encoded polypeptide comprisesor consists of a fragment of Norovirus capsid protein VP1 or a variantof such a fragment. Preferably, the at least one encoded polypeptidecomprises or consists of a C-terminal fragment of Norovirus capsidprotein VP1, or a variant of such a fragment. In another embodiment, theat least one encoded polypeptide comprises or consists of an N-terminalfragment of Norovirus capsid protein VP1, or a variant of such afragment.

Preferably, the at least one encoded polypeptide comprises or consistsof a fragment, preferably a C-terminal fragment, or a variant of such afragment, of a Norovirus capsid protein VP1 as present in a Noroviruspolyprotein (precursor protein) before cleavage. In the context of thepresent invention, the phrase “Norovirus capsid protein VP1 as presentin a Norovirus polyprotein before cleavage” typically refers to acontinuous amino acid sequence beginning at the N-terminus of aNorovirus polyprotein (before cleavage) and comprising the amino acidresidue immediately N-terminal of the first amino acid residue of aprecursor of Norovirus pr protein as present in the Noroviruspolyprotein. In other words, the phrase “Norovirus capsid protein VP1 aspresent in a Norovirus polyprotein before cleavage” may refer to a partof a Norovirus polyprotein corresponding to Norovirus capsid protein VP1comprising a C-terminal fragment, preferably a C-terminal signalsequence, which is typically not present in mature Norovirus proteinVP1. For example, a ‘Norovirus capsid protein VP1 as present in aNorovirus polyprotein before cleavage’ as used herein may comprise anamino acid sequence derived from an amino acid sequence corresponding toamino acid residues 1 to 122 of a Norovirus polyprotein before cleavage.According to a preferred embodiment, a Norovirus capsid protein VP1 aspresent in a Norovirus polyprotein before cleavage comprises an aminoacid sequence according to any one of SEQ ID NOs: 1-4410, or a fragmentor variant of any of these sequences.

Hence, a “C-terminal fragment, or a variant of such a fragment, ofNorovirus capsid protein VP1 as present in a Norovirus polyprotein(precursor protein) before cleavage” preferably comprises an amino acidsequence corresponding to a continuous amino acid sequence, which islocated immediately N-terminal of Norovirus pr protein in a Noroviruspolyprotein before cleavage, or to a fragment or variant of said aminoacid sequence. Preferably, the C-terminal fragment, or a variant of sucha fragment, of Norovirus capsid protein VP1 as present in a Noroviruspolyprotein (precursor protein) before cleavage comprises or consists ofat least 3, 4, 5, 8, 7, 8, 9, or, most preferably, at least 10 aminoacid residues. Alternatively, the C-terminal fragment, or a variant ofsuch a fragment, of Norovirus capsid protein VP1 as present in aNorovirus polyprotein (precursor protein) before cleavage may consist of3 to 40, 3 to 30, 3 to 20, 5 to 20 or 10 to 20 amino acid residues.

According to a preferred embodiment, the at least one polypeptideencoded by the at least one coding region of the inventive artificialnucleic acid comprises or consists of at least one amino acid sequencederived from a signal sequence, or a fragment or variant thereof.

As used herein, the term “signal sequence” preferably refers to an aminoacid sequence, which is involved in the targeting of a protein, e.g. aNorovirus protein, to a cellular compartment, preferably a membrane,more preferably a membrane of the endoplasmic reticulum (ER). A signalsequence in the context of the present invention preferably comprisesfrom 3 to 40, 3 to 30, 3 to 20, 5 to 20 or 10 to 20 amino acid residues.Such a signal sequence may be present, for example, in a Noroviruspolyprotein and may be removed during processing of said polyprotein. Asignal sequence is preferably no longer present in a mature Norovirusprotein. For example, Norovirus capsid protein VP1 as present in aNorovirus polyprotein typically comprises a C-terminal signal sequence,corresponding to the amino acid sequence immediately N-terminal ofNorovirus pr protein (e.g. amino acid residues 105 to 122 in a Noroviruspolyprotein before cleavage). That signal sequence is involved intargeting Norovirus capsid protein VP1 to the ER membrane and istypically removed in order to yield mature Norovirus capsid protein VP1,which no longer comprises said C-terminal fragment comprising a signalsequence.

Preferably, the amino acid sequence derived from a signal sequence, or afragment or variant thereof, comprises at least 3, 4, 5, 8, 7, 8, 9, 10,11, 12, 13, 14, 15, 18, 17, 18, 19 or at least 20 amino acid residues.Alternatively, the amino acid sequence derived from a signal sequence,or a fragment or variant thereof may consist of 3 to 40, 3 to 30, 3 to20, 5 to 20 or 10 to 20 amino acid residues. Most preferably, the aminoacid sequence derived from a signal sequence, or a fragment or variantthereof consists of from 3 to 20 amino acid residues.

In a preferred embodiment, the at least one polypeptide encoded by theat least one coding region of the inventive artificial nucleic acidcomprises or consists of at least one amino acid sequence derived from asignal sequence, which comprises or consists of an amino acid sequencethat is bound by signal recognition particle (SRP). More preferably, theat least one amino acid sequence derived from a signal sequencecomprises or consists of an amino acid sequence that is recognized bysignal peptide peptidase (SPP), by a viral protease and/or by furin or afurin-like protease. Most preferably, the at least one amino acidsequence derived from a signal sequence comprises an amino acid sequencethat is recognized by a viral protease comprising one or more of aNorovirus non-structural protein selected from the group consisting ofNS1/NS2, NS3, NS4, NS5, NS6, and NS7.

In a preferred embodiment, the at least one polypeptide encoded by theat least one coding region of the inventive artificial nucleic acidcomprises or consists of at least one amino acid sequence derived from asignal sequence of a secretory protein or from a signal sequence of amembrane protein. More preferably, the at least one amino acid sequencederived from a signal sequence, preferably derived from a signalsequence of a membrane protein, targets the at least one encoded proteinto a cellular compartment, preferably to the endoplasmic reticulum (ER),more preferably to the ER membrane.

It is further preferred that the at least one polypeptide encoded by theat least one coding region of the inventive artificial nucleic acidcomprises or consists of an amino acid sequence corresponding to asignal sequence from a Norovirus protein, preferably from Noroviruscapsid protein VP1, more preferably from Norovirus capsid protein VP1 aspresent in a Norovirus polyprotein before cleavage, or a fragment orvariant of any of these.

According to a preferred embodiment, the at least one polypeptideencoded by the at least one coding region of the inventive artificialnucleic acid comprises or consists of an amino acid sequencecorresponding to a signal sequence from Norovirus capsid protein VP1 aspresent in a Norovirus polyprotein before cleavage, or a fragment orvariant thereof, wherein the signal sequence is preferably derived froma C-terminal fragment of Norovirus capsid protein VP1 as present in aNorovirus polyprotein before cleavage, preferably as described herein.

According to another preferred embodiment, the at least one polypeptideencoded by the at least one coding region of the inventive artificialnucleic acid comprises or consists of a fragment, preferably aC-terminal fragment, or a variant of such a fragment, of a matureNorovirus protein, preferably of a mature Norovirus capsid protein VP1.In this context, it is preferred that the mature Norovirus protein is amature Norovirus protein as defined herein.

According to a preferred embodiment, the at least one polypeptideencoded by the at least one coding region of the inventive artificialnucleic acid comprises or consists of a fragment, preferably aC-terminal fragment, or a variant of such a fragment, of matureNorovirus capsid protein VP1, wherein the mature Norovirus capsidprotein VP1 does preferably not comprise a C-terminal signal sequence asdescribed herein with respect to a Norovirus capsid protein VP1 aspresent in a Norovirus polyprotein (before cleavage). More preferably,the mature Norovirus capsid protein VP1 comprises or consists of anamino acid sequence according to any one of SEQ ID NOs: 1-4410, or afragment or variant thereof.

Preferably, the at least one polypeptide encoded by the at least onecoding region of the inventive artificial nucleic acid comprises orconsists of a C-terminal fragment, preferably as defined herein, or avariant of such a fragment, of mature Norovirus capsid protein VP1.

Preferably, the C-terminal fragment, or a variant of such a fragment, ofmature Norovirus capsid protein VP1 comprises or consists of at least 3,4, 5, 8, 7, 8, 9, or, most preferably, at least 10 amino acid residues.Alternatively, the C-terminal fragment, or a variant of such a fragment,of mature Norovirus capsid protein VP1 may comprise or consist of 3 to40, 3 to 30, 3 to 20, 3 to 10, 5 to 20 or 10 to 20 amino acid residues.

According to another embodiment, the at least one polypeptide encoded bythe at least one coding region of the inventive artificial nucleic acidcomprises or consists of an amino acid sequence derived from a

-   -   a) a C-terminal fragment, or a variant of such a fragment, of a        mature Norovirus capsid protein VP1, preferably as defined        herein    -   and    -   b) a C-terminal fragment, or a variant of such a fragment, of        Norovirus capsid protein VP1 as present in a Norovirus        polyprotein (precursor protein) before cleavage, preferably as        defined herein; or a signal sequence, or a fragment or variant        thereof, preferably as defined herein.

Therein, the amino acid sequence according to a) may be in continuationwith the amino acid sequence according to b), wherein the sequences maybe positioned relative to each other in any manner. Alternatively, theamino acid sequences according to a) and b) may be separated in the atleast one encoded protein by another amino acid sequence. Mostpreferably, the amino acid sequence according to a) is locatedN-terminally with respect to b).

According to a preferred embodiment, the at least one polypeptideencoded by the at least one coding region of the inventive artificialnucleic acid comprises or consists of at least one amino acid sequencecorresponding to a fragment of Norovirus non-structural protein 1(NS1/2), or a variant of such a fragment.

As used herein, the term “fragment of Norovirus non-structural protein 1(NS1/2)” preferably relates to a continuous amino acid sequence derivedfrom Norovirus non-structural protein 1 (NS1/2), or to a fragment orvariant of said continuous amino acid sequence.

Preferably, the fragment, or variant thereof, of Norovirusnon-structural protein 1 (NS1/2) comprises or consists of at least 3, 4,5, 8, 7, 8, 9, or, most preferably, at least 10 amino acid residues.Alternatively, the fragment, or variant thereof, of Norovirusnon-structural protein 1 (NS1/2) may comprise or consist of 3 to 40, 3to 30, 3 to 20, 3 to 10, 5 to 20 or 10 to 20 amino acid residues. Mostpreferably, the fragment, or variant thereof, of Norovirusnon-structural protein 1 (NS1/2) comprises or consists of from 3 to 20amino acid residues.

In a preferred embodiment, the at least one polypeptide encoded by theat least one coding region of the inventive artificial nucleic acidcomprises or consists of at least one amino acid sequence correspondingto an N-terminal fragment of Norovirus non-structural protein 1 (NS1/2),or a variant of said fragment.

In the context of the present invention, the term “N-terminal fragmentof Norovirus non-structural protein 1 (NS1/2)” relates to a continuousamino acid sequence derived from the N-terminus of Norovirusnon-structural protein 1 (NS1/2). More preferably, the N-terminalfragment of Norovirus non-structural protein 1 (NS1/2) comprises orconsists of from 3 to 20 amino acid residues. In a preferred embodiment,the at least one encoded polypeptide comprises an N-terminal fragment ofNorovirus non-structural protein 1 (NS1/2), wherein the N-terminalfragment of Norovirus non-structural protein 1 (NS1/2) is a continuousamino acid sequence comprising or consisting of 3 to 20 amino acidresidues corresponding to a continuous amino acid sequence of 3 to 20amino acid residues in the first 20 amino acid residues (counting fromthe N-terminus) of Norovirus non-structural protein 1 (NS1/2), or avariant thereof.

In one embodiment, the at least one encoded polypeptide comprises orconsists of an N-terminal fragment of Norovirus non-structural protein 1(NS1/2), wherein the N-terminal fragment of Norovirus non-structuralprotein 1 (NS1/2) is a continuous amino acid sequence comprising orconsisting of 3 to 20 amino acid residues corresponding to a continuousamino acid sequence of 3 to 20 amino acid residues in the first 20 aminoacid residues (counting from the N-terminus) of a mature Norovirusnon-structural protein 1 (NS1/2), or a variant thereof. Therein, thefirst 20 amino acid residues of a mature Norovirus non-structuralprotein 1 (NS1/2) preferably comprise or consist of the N-terminusitself (i.e. the amino acid residue at the N-terminus) and the 19following amino acid residues.

In a preferred embodiment, the at least one polypeptide encoded by theat least one coding region of the inventive artificial nucleic acidcomprises a first Norovirus protein, which is preferably a Norovirusprotein as described herein, or a fragment or variant thereof, andfurther comprises at least one second or further Norovirus protein, or afragment or variant thereof, wherein the at least one second or furtherNorovirus protein, or the fragment or variant thereof, is distinct fromthe first Norovirus protein, or the fragment or variant thereof.

In that embodiment, the first Norovirus protein is preferably selectedfrom the group consisting of Norovirus NS1/NS2, NS32A, NS428, NS53,NS64A, NS4B or NS75, or a fragment or variant thereof. Preferably, thesecond or further Norovirus protein is selected from the groupconsisting of Norovirus capsid protein VP1, Norovirus capsid protein VP2and a Norovirus non-structural protein, preferably Norovirus NS1/NS2,NS3, NS4, NS5, NS6, or NS7, or a fragment or variant thereof.

More preferably, the at least one polypeptide encoded by the at leastone coding region of the inventive artificial nucleic acid comprisesNorovirus capsid protein VP1 and/or VP2, or a fragment or variantthereof, and further comprises at least one of the following:

-   -   a) an amino acid sequence corresponding to a C-terminal        fragment, or a variant thereof, of mature Norovirus capsid        protein VP1, preferably as described herein;    -   b) an amino acid sequence corresponding to a C-terminal        fragment, or a variant thereof, of Norovirus capsid protein VP1        as present in Norovirus polyprotein before cleavage, preferably        as described herein;    -   c) an amino acid sequence corresponding to an N-terminal        fragment, or a variant thereof, of Norovirus non-structural        protein 1 (NS1/NS2), preferably as described herein; and/or    -   d) an amino acid corresponding to a fragment of Norovirus        NS1/NS2, NS3, NS4, NS5, NS6, or NS7.

In a further embodiment, the at least one polypeptide encoded by the atleast one coding region of the inventive artificial nucleic acidcomprises or consists of, preferably in this order from N-terminus toC-terminus, Norovirus capsid protein VP1, or a fragment or variantthereof,

and Norovirus non-structural protein 1 (NS1/NS2), or a fragment orvariant thereof.

According to a preferred embodiment, the inventive artificial nucleicacid is monocistronic, bicistronic or multicistronic.

Preferably, the inventive artificial nucleic acid is monocistronic. Inthat embodiment, the inventive artificial nucleic acid comprises onecoding region, wherein the coding region encodes a polypeptidecomprising at least two different Norovirus proteins, preferably asdefined herein, or a fragment or variant thereof.

Alternatively, the inventive artificial nucleic acid can be bi- ormulticistronic and comprises at least two coding regions, wherein the atleast two coding regions encode at least two polypeptides, wherein eachof the at least two polypeptides comprises at least one differentNorovirus protein, preferably as described herein, or a fragment orvariant of any one of these proteins. For example, the inventiveartificial nucleic acid may comprise two coding regions, wherein thefirst coding region encodes a first polypeptide comprising a firstNorovirus protein, or a fragment or variant thereof, and wherein thesecond coding region encodes a second polypeptide comprising a secondNorovirus protein, or a fragment or variant thereof, wherein the firstand second Norovirus proteins or a fragment or variant thereof aredistinct from each other.

The inventive artificial nucleic acid may be provided as DNA or as RNA,preferably an RNA as defined herein. More preferably, the inventiveartificial nucleic acid is an artificial mRNA.

The inventive artificial nucleic acid may further be single stranded ordouble stranded. When provided as a double stranded nucleic acid, theinventive artificial nucleic acid preferably comprises a sense and acorresponding antisense strand.

Preferably, the inventive artificial nucleic acid as defined hereintypically comprises a length of about 50 to about 20000, or 100 to about20000 nucleotides, preferably of about 250 to about 20000 nucleotides,more preferably of about 500 to about 10000, even more preferably ofabout 500 to about 5000.

Nucleic Acid Modifications:

According to one embodiment, the inventive artificial nucleic acid asdefined herein, may be in the form of a modified nucleic acid,preferably a modified mRNA, wherein any modification, as defined herein,may be introduced into the inventive artificial nucleic acid.Modifications as defined herein preferably lead to a stabilizedartificial nucleic acid, preferably a stabilized artificial RNA, of thepresent invention.

According to one embodiment, the inventive artificial nucleic acid,preferably an mRNA, may thus be provided as a “stabilized nucleic acid”,preferably as a “stabilized mRNA”, that is to say as a nucleic acid,preferably an mRNA, that is essentially resistant to in vivo degradation(e.g. by an exo- or endo-nuclease). Such stabilization can be effected,for example, by a modified phosphate backbone of an artificial mRNA ofthe present invention. A backbone modification in connection with thepresent invention is a modification in which phosphates of the backboneof the nucleotides contained in the mRNA are chemically modified.Nucleotides that may be preferably used in this connection contain e.g.a phosphorothioate-modified phosphate backbone, preferably at least oneof the phosphate oxygens contained in the phosphate backbone beingreplaced by a sulfur atom. Stabilized artificial nucleic acids,preferably mRNAs, may further include, for example: non-ionic phosphateanalogues, such as, for example, alkyl and aryl phosphonates, in whichthe charged phosphonate oxygen is replaced by an alkyl or aryl group, orphosphodiesters and alkylphosphotriesters, in which the charged oxygenresidue is present in alkylated form. Such backbone modificationstypically include, without implying any limitation, modifications fromthe group consisting of methylphosphonates, phosphoramidates andphosphorothioates (e.g. cytidine-5′-0-(1-thiophosphate)).

In the following, specific modifications are described, which arepreferably capable of “stabilizing” the inventive artificial nucleicacid, preferably an mRNA, as defined herein.

Chemical Modifications:

The terms “nucleic acid modification” as used herein may refer tochemical modifications comprising backbone modifications as well assugar modifications or base modifications.

In this context, a modified artificial nucleic acid, preferably an mRNA,as defined herein may contain nucleotide analogues/modifications, e.g.backbone modifications, sugar modifications or base modifications. Abackbone modification in connection with the present invention is amodification, in which phosphates of the backbone of the nucleotidescontained in an artificial nucleic acid, preferably an mRNA, as definedherein are chemically modified. A sugar modification in connection withthe present invention is a chemical modification of the sugar of thenucleotides of the artificial nucleic acid, preferably an mRNA, asdefined herein. Furthermore, a base modification in connection with thepresent invention is a chemical modification of the base moiety of thenucleotides of the artificial nucleic acid, preferably an mRNA. In thiscontext, nucleotide analogues or modifications are preferably selectedfrom nucleotide analogues, which are applicable for transcription and/ortranslation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified artificial nucleic acid, preferably an mRNA, as describedherein, can be modified in the sugar moiety. For example, the 2′hydroxyl group (OH) can be modified or replaced with a number ofdifferent “oxy” or “deoxy” substituents. Examples of “oxy”-2′ hydroxylgroup modifications include, but are not limited to, alkoxy or aryloxy(-DR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR; “locked” nucleic acids(LNA) in which the 2′ hydroxyl is connected, e.g., by a methylenebridge, to the 4′ carbon of the same ribose sugar; and amino groups(—O-amino, wherein the amino group, e.g., NRR, can be alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroaryl amino, ethylene diamine, polyamine) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, an artificial nucleic acid, preferably an mRNA,can include nucleotides containing, for instance, arabinose as thesugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into a modifiedartificial nucleic acid, preferably an mRNA, as described herein. Thephosphate groups of the backbone can be modified by replacing one ormore of the oxygen atoms with a different substituent. Further, themodified nucleosides and nucleotides can include the full replacement ofan unmodified phosphate moiety with a modified phosphate as describedherein. Examples of modified phosphate groups include, but are notlimited to, phosphorothioate, phosphoroselenates, borano phosphates,borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkylor aryl phosphonates and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bemodified by the replacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified nucleic acid, preferably an mRNA, as described herein canfurther be modified in the nucleobase moiety. Examples of nucleobasesfound in a nucleic acid such as RNA include, but are not limited to,adenine, guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-G-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiacytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-D-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bramouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5¹-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5¹-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5¹-triphosphate,6-chloropurineriboside-5¹-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5¹-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5¹-triphosphate, 7-deazaguanosine-5¹-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.In other embodiments, modified nucleosides include 2-aminopurine, 2,G-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group. In specific embodiments, a modified nucleoside is5′-0-(1-thiophosphate)-adenosine, 5′-0-(1-thiophosphate)-cytidine,5′-0-(1-thiophosphate)-guanosine, 5′-0-(1-thiophosphate)-uridine or5′-0-(1-thiophosphate)-pseudouridine.

In further specific embodiments, a modified artificial nucleic acid,preferably an mRNA, may comprise nucleoside modifications selected from6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, Pseudo-iso-cytidine,5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine,5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine,5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine,inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, α-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

Lipid Modification:

According to a further embodiment, a modified artificial nucleic acid,preferably an mRNA, as defined herein can contain a lipid modification.Such a lipid-modified artificial nucleic acid as defined hereintypically further comprises at least one linker covalently linked withthat artificial nucleic acid, and at least one lipid covalently linkedwith the respective linker. Alternatively, the lipid-modified artificialnucleic acid comprises at least one artificial nucleic acid as definedherein and at least one (bifunctional) lipid covalently linked (withouta linker) with that artificial nucleic acid. According to a thirdalternative, the lipid-modified artificial nucleic acid comprises anartificial nucleic acid molecule as defined herein, at least one linkercovalently linked with that artificial nucleic acid, and at least onelipid covalently linked with the respective linker, and also at leastone (bifunctional) lipid covalently linked (without a linker) with thatartificial nucleic acid. In this context, it is particularly preferredthat the lipid modification is present at the terminal ends of a linearartificial nucleic acid.

Coding Sequence Modifications:

G/C Content Modification:

According to another embodiment, the artificial nucleic acid of thepresent invention may be modified, and thus stabilized, by modifying theG/C content of the artificial nucleic acid, preferably an mRNA,preferably of the coding region of the inventive artificial nucleicacid.

Preferably, the G/C content of the at least one coding region of theartificial nucleic acid, preferably an mRNA, is modified, preferablyincreased, compared to the G/C content of the corresponding codingsequence of the wild type nucleic acid, preferably an mRNA, wherein theencoded amino acid sequence is preferably not modified compared to theamino acid sequence encoded by the corresponding wild type nucleic acid(i.e. the non-modified nucleic acid), preferably an mRNA. Thismodification of the inventive artificial nucleic acid, preferably of anmRNA, as described herein is based on the fact that the sequence of anymRNA region to be translated is important for efficient translation ofthat mRNA. Thus, the composition and the sequence of various nucleotidesare important. In particular, sequences having an increased G(guanosine)/C (cytosine) content are more stable than sequences havingan increased A (adenosine)/U (uracil) content. According to theinvention, the codons of the artificial nucleic acid, preferably anmRNA, are therefore varied compared to the respective wild type mRNA,while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons encode one and the same amino acid (so-calleddegeneration of the genetic code), the most favorable codons for thestability can be determined (so-called alternative codon usage).Depending on the amino acid to be encoded by the artificial nucleicacid, preferably an mRNA, there are various possibilities formodification of its sequence, compared to its wild type sequence. In thecase of amino acids which are encoded by codons, which containexclusively G or C nucleotides, no modification of the codon isnecessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala(GCC or GCG) and Gly (GGC or GGG) require no modification, since no A orU is present. In contrast, codons which contain A and/or U nucleotidescan be modified by substitution of other codons, which code for the sameamino acids but contain no A and/or U. Examples of these are: the codonsfor Pro can be modified from CCU or CCA to CCC or CCG; the codons forArg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; thecodons for Ala can be modified from GCU or GCA to GCC or GCG; the codonsfor Gly can be modified from GGU or GGA to GGC or GGG. In other cases,although A or U nucleotides cannot be eliminated from the codons, it ishowever possible to decrease the A and U content by using codons, whichcontain a lower content of A and/or U nucleotides. Examples of theseare: the codons for Phe can be modified from UUU to UUC; the codons forLeu can be modified from UUA, UUG, CUU or CUA to CUC or CUG the codonsfor Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC; thecodon for Tyr can be modified from UAU to UAC; the codon for Cys can bemodified from UGU to UGC; the codon for His can be modified from CAU toCAC; the codon for Gln can be modified from CAA to CAG; the codons forIle can be modified from AUU or AUA to AUC; the codons for Thr can bemodified from ACU or ACA to ACC or ACG; the codon for Asn can bemodified from AAU to AAC; the codon for Lys can be modified from AAA toAAG; the codons for Val can be modified from GUU or GUA to GUC or GUG;the codon for Asp can be modified from GAU to GAC; the codon for Glu canbe modified from GAA to GAG; the stop codon UAA can be modified to uAGor UGA. In the case of the codons for Met (AUG) and Trp (UGG), on theother hand, there is no possibility of sequence modification. Thesubstitutions listed above can be used either individually or in allpossible combinations to increase the G/C content of the inventiveartificial nucleic acid, preferably an mRNA, compared to itscorresponding wild type sequence, such as the corresponding wild typemRNA sequence. Thus, for example, all codons for Thr occurring in thewild type sequence can be modified to ACC (or ACG). Preferably, however,for example, combinations of the above substitution possibilities areused:

substitution of all codons coding for Thr in the original sequence (wildtype mRNA) to ACC (or ACG) andsubstitution of all codons originally coding for Ser to UCC (or UCG orADC); substitution of all codons coding for Ile in the original sequenceto AUG and substitution of all codons originally coding for Lys to AAGand substitution of all codons originally coding for Tyr to DC;substitution of all mhos coding for Val in the original sequence to DUD(or GUG) and substitution of all codons originally coding for Glu to GAGand substitution of all codons originally coding for Ala to GCC (or GCG)and substitution of all codons originally coding for Arg to CGC (orCGG); substitution of all codons coding for Val in the original sequenceto GUC (or GUG) and substitution of all codons originally coding for Gluto GAB and substitution of all codons originally coding for Ala to GCC(or GCG) and substitution of all codons originally coding for Gly to GGC(or GGG) and substitution of all codons originally coding for Asn toAAC; substitution of all codons coding for Val in the original sequenceto GUC (or GUG) and substitution of all codons originally coding for Pheto UUC and substitution of all codons originally coding for Cys to UGCand substitution of all codons originally coding for Leu to CUG (or CUC)and substitution of all codons originally coding for Gln to CAG andsubstitution of all codons originally coding for Pro to CCC (or CCG);etc. Preferably, the G/C content of the coding region of the inventiveartificial nucleic acid, preferably an mRNA, is increased by at least7%, more preferably by at least 15%, particularly preferably by at least20%, compared to the G/C content of the coding region of the wild typenucleic acid. According to a specific embodiment at least 5%, 10%, 20%,30%, 40%, 50%, 60%, more preferably at least 70%, even more preferablyat least 80% and mast preferably at least 90%, 95% or even 100% of thesubstitutable codons in the coding region or the whale sequence of thewild type nucleic acid sequence, preferably an mRNA sequence, aresubstituted, thereby increasing the G/C content of said sequence. Inthis context, it is particularly preferable to increase the G/C contentof the inventive artificial nucleic acid to the maximum (i.e. 100% ofthe substitutable codons), in particular in the region coding for the atleast one protein, compared to the wild type sequence.

In one embodiment of the invention, the G/C content of the artificialnucleic acid of the invention is increased compared to the G/C contentof the corresponding coding sequence of the wild type mRNA, or whereinthe C content of the coding region of the mRNA sequence is increasedcompared to the C content of the corresponding coding sequence of thewild type mRNA, or wherein the codon usage in the coding region of themRNA sequence is adapted to the human codon usage, or wherein the codonadaptation index (CAI) is increased or maximised in the coding region ofthe mRNA sequence, wherein the encoded amino acid sequence of the mRNAsequence is preferably not being modified compared to the encoded aminoacid sequence of the wild type mRNA.

In a further embodiment, the artificial nucleic acid according to theinvention is codon optimized, wherein

-   -   (i) the at least one coding region comprises a nucleic acid        sequence, which is codon-optimized; and/or    -   (ii) the at least one coding sequence comprises a nucleic acid        sequence, which is identical or at least 50%, 60%, 70%, 80%,        85%, 89%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a nucleic acid sequence selected from        the group consisting of SEQ ID NOs: 8821-13230, 26461-39690,        39715, 39716, 39717, 39720, 39721, 39724, 39725, 39728, 39729,        39730, 39733, 39734, 39737, 39738, 39741, 39742, 39745 and        39746, or a fragment or variant of any of these sequences;        and/or    -   (iii) the at least one coding sequence comprises a nucleic acid        sequence, which is identical or at least 50%, 60%, 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a nucleic acid sequence selected from        the group consisting of SEQ ID NOs: 13231-17640, or a fragment        or variant of any of these sequences; and/or    -   (iv) the artificial nucleic acid of the invention, wherein the        at least one coding sequence comprises a nucleic acid sequence,        which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to a nucleic acid sequence selected from the group        consisting of SEQ ID NOs: 17641-22050, or a fragment or variant        of any of these sequences; and/or    -   (v) the artificial nucleic acid of the invention, wherein the at        least one coding sequence comprises a nucleic acid sequence,        which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to a nucleic acid sequence selected from the group        consisting of SEQ ID NOs: 22051-26460, or a fragment or variant        of any of these sequences.

According to the invention, a further preferred modification of theartificial nucleic acid of the present invention is based on the findingthat the translation efficiency is also determined by a differentfrequency in the occurrence of tRNAs in cells. It is thus preferred thatthe at least one coding region of the artificial nucleic acid accordingto the invention comprises a nucleic acid sequence, which iscodon-optimized. The term “codon-optimized” as used herein typicallyrefers to an artificial nucleic acid, preferably to a nucleic acidsequence in the at least one coding region therein, wherein at least oneradon of the wild type sequence, which codes for a tRNA which isrelatively rare in the cell, is exchanged for a radon, which codes for atRNA which is relatively frequent in the cell and carries the same aminoacid as the relatively rare tRNA. Most preferably, that modificationalso increases the G/C content of the at least one coding region of theartificial nucleic acid.

Thus, if so-called “rare codons” are present in the artificial nucleicacid of the present invention to an increased extent, the correspondingmodified nucleic acid sequence, preferably an mRNA sequence, istranslated to a significantly poorer degree than in the case wherecodons coding for relatively “frequent” tRNAs are present. According tothe invention, in the modified artificial nucleic acid of the presentinvention, the region which encodes the at least one protein as definedherein is modified compared to the corresponding region of the wild typenucleic acid, preferably an mRNA, such that at least one radon of thewild type sequence, which codes for a tRNA which is relatively rare inthe cell, is exchanged for a radon, which codes for a tRNA which isrelatively frequent in the cell and carries the same amino acid as therelatively rare tRNA. By this modification, the sequences of theartificial nucleic acid of the present invention is modified such thatcodons for which frequently occurring tRNAs are available are inserted.In other words, according to the invention, by this modification allcodons of the wild type sequence which code for a tRNA which isrelatively rare in the cell can in each case be exchanged for a codonwhich codes for a tRNA which is relatively frequent in the cell andwhich, in each case, carries the same amino acid as the relatively raretRNA. Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly radon, which uses the tRNA, which occursthe most frequently in the (human) cell, are particularly preferred.According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified artificial nucleic acid of the present invention, with the“frequent” codons without modifying the amino acid sequence of theprotein encoded by the coding region of the corresponding wild typenucleic acid, preferably an mRNA. This preferred embodiment allowsprovision of a particularly efficiently translated and stabilized(modified) artificial nucleic acid of the present invention. Thedetermination of an artificial nucleic acid of the present invention asdescribed above (increased G/C content; exchange of tRNAs) can becarried out using the computer program explained in WO 02/098443—thedisclosure content of which is included in its full scope in the presentinvention. Using this computer program, the nucleotide sequence of anydesired mRNA can be modified with the aid of the genetic code or thedegenerative nature thereof such that a maximum G/C content results, incombination with the use of codons which code for tRNAs occurring asfrequently as possible in the cell, the amino acid sequence encoded bythe artificial nucleic acid preferably not being modified compared tothe non-modified sequence. Alternatively, it is also possible to modifyonly the G/C content or only the radon usage compared to the originalsequence. The source code in Visual Basic 6.0 (development environmentused: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is alsodescribed in WO 02/098443. In a further preferred embodiment of thepresent invention, the A/U content in the environment of the ribosomebinding site of the artificial nucleic acid of the present invention isincreased compared to the A/U content in the environment of the ribosomebinding site of its particular wild type nucleic acid, preferably anmRWA. This modification (an increased A/U content around the ribosomebinding site) increases the efficiency of ribosome binding to theartificial nucleic acid. An effective binding of the ribosomes to theribosome binding site (e.g. a Kozak sequence as known in the art) inturn has the effect of an efficient translation of the artificialnucleic acid. According to a further embodiment of the presentinvention, the artificial nucleic acid of the present invention may bemodified with respect to potentially destabilizing sequence elements.Particularly, the coding region and/or the 5′ and/or 3′ untranslatedregion of the artificial nucleic acid may be modified compared to theparticular wild type nucleic acid such that it contains no destabilizingsequence elements, the amino acid sequence encoded by the modifiedartificial nucleic acid preferably not being modified compared to itsparticular wild type nucleic acid. It is known that, for example, insequences of eukaryotic RNAs destabilizing sequence elements (USE)occur, to which signal proteins bind and regulate enzymatic degradationof RNA in vivo. For further stabilization of the modified artificialnucleic acid, optionally in the region which encodes the at least oneprotein as defined herein, one or more such modifications compared tothe corresponding region of the wild type nucleic acid, preferably anmRWA, can therefore be carried out, so that no or substantially nodestabilizing sequence elements are contained there. According to theinvention, DSE present in the untranslated regions (3′- and/or 5′-UTR)can also be eliminated from the artificial nucleic acid of the presentinvention by such modifications. Such destabilizing sequences are e.g.AU-rich sequences (AURES), which occur in 3′-UTR sections of numerousunstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83:1670 to1674). The artificial nucleic acid of the present invention is thereforepreferably modified compared to the wild type nucleic acid such that theartificial nucleic acid contains no such destabilizing sequences. Thisalso applies to those sequence motifs which are recognized by possibleendonucleases, e.g. the sequence GAACAAG, which is contained in the3′-UTR segment of the gene which codes for the transferrin receptor(Binder et al., EMBO J. 1994, 13: 1999 to 1980). These sequence motifsare also preferably removed in the artificial nucleic acid of thepresent invention. It is further preferred that the artificial nucleicacid of the present invention has, in a modified form, at least one IRESas defined above and/or at least one 5′ and/or 3′ stabilizing sequence,in a modified form, e.g. to enhance ribosome binding or to allowexpression of different encoded polypeptides located on an artificialnucleic acid of the present invention. This particularly applies toembodiments, wherein the artificial nucleic acid is bi- ormulticistronic and wherein an IRES is preferably located betweenindividual coding regions.

According to a preferred embodiment, the at least one coding region ofthe artificial nucleic acid or artificial nucleic acid moleculecomprises or consists of at least one nucleic acid sequence according toany one of SEQ ID NOs: 8821-39690, 39715, 39719, 39717, 39720, 39721,39724, 39725, 39728, 39729, 39730, 39733, 39734, 39737, 39738, 39741,39742, 39745 and 39749, or a fragment or variant of any of thesesequences. More preferably, the at least one coding region of theartificial nucleic comprises or consists of an RNA sequence, which is atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 8821-39990, 39715, 39719, 39717, 39720, 39721, 39724, 39725,39728, 39729, 39730, 39733, 39734, 39737, 39738, 39741, 39742, 39745 and39749.

G/C Content Modification:

According to another embodiment, the RNA of the present invention,preferably an mRNA, may be modified, and thus stabilized, by modifyingthe guanosine/cytosine (G/C) content of the RNA, preferably of the atleast one coding sequence of the RNA of the present invention.

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region of the RNA of the present invention ismodified, particularly increased, compared to the G/C content of thecoding region of the respective wild type RNA, i.e. the unmodified RNA.The amino acid sequence encoded by the RNA is preferably not modified ascompared to the amino acid sequence encoded by the respective wild typeRNA. This modification of the RNA of the present invention is based onthe fact that the sequence of any RNA region to be translated isimportant for efficient translation of that RNA. Thus, the compositionof the RNA and the sequence of various nucleotides are important. Inparticular, sequences having an increased G (guanosine)/C (cytosine)content are more stable than sequences having an increased A(adenosine)/U (uracil) content. According to the invention, the codonsof the RNA are therefore varied compared to the respective wild typeRNA, while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favourable codons for thestability can be determined (so-called alternative codon usage).Depending on the amino acid to be encoded by the RNA, there are variouspossibilities for modification of the RNA sequence, compared to its wildtype sequence. In the case of amino acids, which are encoded by codons,which contain exclusively G or C nucleotides, no modification of thecodon is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC orCGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification,since no A or U is present. In contrast, codons which contain A and/or Unucleotides can be modified by substitution of other codons, which codefar the same amino acids but contain no A and/or U. Examples of theseare: the codons for Pro can be modified from CCU or CCA to CCC or CCG;the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGCor CGG; the codons for Ala can be modified from GGU or GCC to GCC orGCG; the codons for Gly can be modified from GGU or GGA to GGC or GGG.In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons which contain a lower content of A and/or U nucleotides.Examples of these are: the codons for Phe can be modified from UUU toUUC; the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUCor CUG; the codons for Ser can be modified from UCU or UCA or AGU toUCC, UCG or AGC; the codon for Tyr can be modified from UAU to UAC; thecodon for Cys can be modified from UGU to UGC; the codon for His can bemodified from CAU to CAC; the codon for Gln can be modified from CAA toCAG; the codons for Ile can be modified from AUU or AUA to AUC; thecodons for Thr can be modified from ACU or ACA to ACC or ACG; the codonfor Asn can be modified from AAU to AAC; the codon for Lys can bemodified from AAA to MG; the codons for Val can be modified from GUU orGUA to GUC or GUG; the codon for Asp can be modified from GAU to GAC;the codon for Glu can be modified from GAA to GAG; the stop codon UAAcan be modified to UAG or UGA. In the case of the codons for Met (AUG)and Trp (UGG), on the other hand, there is no possibility of sequencemodification. The substitutions listed above can be used eitherindividually or in all possible combinations to increase the G/C contentof the at least one mRNA of the composition of the present inventioncompared to its particular wild type mRNA (i.e. the original sequence).Thus, for example, all codons for Thr occurring in the wild typesequence can be modified to ACC (or ACG). Preferably, however, forexample, combinations of the above substitution possibilities are used:

substitution of all codons coding for Thr in the original sequence (wildtype mRNA) to ACC (or AGG) and substitution of all codons originallycoding for Ser to UCC (or UCG or AGC); substitution of all codons codingfor Ile in the original sequence to AUC and substitution of all codonsoriginally coding for Lys to AAG and substitution of all codonsoriginally coding for Tyr to UAC; substitution of all codons coding forVal in the original sequence to GUC (or GUG) and substitution of allcodons originally coding for Glu to GAG andsubstitution of all codons originally coding for Ala to GCC (or GCG) andsubstitution of all codons originally coding for Arg to CGC (or CGG);substitution of all codons coding for Val in the original sequence toGUC (or GUG) and substitution of all codons originally coding for Glu toGAG and substitution of all codons originally coding for Ala to GCC (orGCG) and substitution of all codons originally coding for Gly to GGC (orGGG) and substitution of all codons originally coding for Asn to AAC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) and substitution of all codons originally coding for Phe toUUC and substitution of all codons originally coding for Cys to UGC andsubstitution of all codons originally coding for Leu to CUG (or CUC) andsubstitution of all codons originally coding for Gln to CAG andsubstitution of all codons originally coding for Pro to CCC (or CCG);etc.

Preferably, the G/C content of the coding region of the RNA of thepresent invention is increased by at least 7%, more preferably by atleast 15%, particularly preferably by at least 20%, compared to the G/Ccontent of the coding region of the wild type RNA, which codes for anantigen as defined herein or a fragment or variant thereof. According toa specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, morepreferably at least 70%, even more preferably at least 80% and mostpreferably at least 90%, 95% or even 100% of the substitutable codons inthe region coding for an antigen as defined herein or a fragment orvariant thereof or the whole sequence of the wild type RNA sequence aresubstituted, thereby increasing the GC/content of said sequence. In thiscontext, it is particularly preferable to increase the G/C content ofthe RNA of the present invention, preferably of the at least one codingregion of the RNA according to the invention, to the maximum (i.e. 100%of the substitutable codons) as compared to the wild type sequence.According to the invention, a further preferred modification of the RNAof the present invention is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in theRNA of the present invention to an increased extent, the correspondingmodified RNA sequence is translated to a significantly poorer degreethan in the case where codons coding for relatively “frequent” tRNAs arepresent. According to the invention, in the modified RNA of the presentinvention, the region which codes for an antigen as defined herein or afragment or variant thereof is modified compared to the correspondingregion of the wild type RNA such that at least one codon of the wildtype sequence, which codes for a tRNA which is relatively rare in thecell, is exchanged for a codon, which codes for a tRNA which isrelatively frequent in the cell and carries the same amino acid as therelatively rare tRNA. By this modification, the sequences of the RNA ofthe present invention are modified such that codons for which frequentlyoccurring tRNAs are available are inserted. In other words, according tothe invention, by this modification all codons of the wild typesequence, which code for a tRNA which is relatively rare in the cell,can in each case be exchanged for a codon, which codes for a tRNA whichis relatively frequent in the cell and which, in each case, carries thesame amino acid as the relatively rare tRNA. Which tRNAs occurrelatively frequently in the cell and which, in contrast, occurrelatively rarely is known to a person skilled in the art; cf. e.g.Akashi, Curr. Opin. Genet. Dev. 2001, 11(9): 690-999. The codons, whichuse for the particular amino acid the tRNA which occurs the mostfrequently, e.g. the Gly codon, which uses the tRNA, which occurs themast frequently in the (human) cell, are particularly preferred.According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified RNA of the present invention, with the “frequent” codonswithout modifying the amino acid sequence of the protein encoded by thecoding region of the RNA. This preferred embodiment allows provision ofa particularly efficiently translated and stabilized (modified) RNA ofthe present invention. The determination of a modified RNA of thepresent invention as described above (increased G/C content; exchange oftRNAs) can be carried out using the computer program explained in WO02/098443 the disclosure content of which is included in its full scopein the present invention. Using this computer program, the nucleotidesequence of any desired RNA can be modified with the aid of the geneticcode or the degenerative nature thereof such that a maximum G/C contentresults, in combination with the use of codons which code for tRNAsoccurring as frequently as possible in the cell, the amino acid sequencecoded by the modified RNA preferably not being modified compared to thenon-modified sequence. Alternatively, it is also possible to modify onlythe G/C content or only the codon usage compared to the originalsequence. The source code in Visual Basic 6.0 (development environmentused: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is alsodescribed in WO 02/098443. In a further preferred embodiment of thepresent invention, the A/U content in the environment of the ribosomebinding site of the RNA of the present invention is increased comparedto the A/U content in the environment of the ribosome binding site ofits respective wild type mRNA. This modification (an increased A/Ucontent around the ribosome binding site) increases the efficiency ofribosome binding to the RNA. An effective binding of the ribosomes tothe ribosome binding site (Kozak sequence: SEQ ID NOs: 39711, 39712; theAUG forms the start codon) in turn has the effect of an efficienttranslation of the RNA. According to a further embodiment of the presentinvention, the RNA of the present invention may be modified with respectto potentially destabilizing sequence elements. Particularly, the codingregion and/or the 5′ and/or 3′ untranslated region of this RNA may bemodified compared to the respective wild type RNA such that it containsno destabilizing sequence elements, the encoded amino acid sequence ofthe modified RNA preferably not being modified compared to itsrespective wild type RNA. It is known that, for example in sequences ofeukaryotic RNAs, destabilizing sequence elements (DSE) occur, to whichsignal proteins bind and regulate enzymatic degradation of RNA in vivo.For further stabilization of the modified RNA, optionally in the regionwhich encodes an antigen as defined herein or a fragment or variantthereof, one or more such modifications compared to the correspondingregion of the wild type RNA can therefore be carried out, so that no orsubstantially no destabilizing sequence elements are contained there.According to the invention, DSE present in the untranslated regions (3′-and/or 5′-UTR) can also be eliminated from the RNA of the presentinvention by such modifications. Such destabilizing sequences are e.g.AU-rich sequences (AURES), which occur in 3′-UTR sections of numerousunstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83:1670 to1674). The RNA of the present invention is therefore preferably modifiedcompared to the respective wild type RNA such that the RNA of thepresent invention contains no such destabilizing sequences. This alsoapplies to those sequence motifs which are recognized by possibleendonucleases, e.g. the sequence GAACAAG, which is contained in the3′-UTR segment of the gene encoding the transferrin receptor (Binder etal., EMBO J. 1994, 13: 1969 to 1980). These sequence motifs are alsopreferably removed in the RNA of the present invention.

According to a preferred embodiment, the present invention provides anRNA as defined herein comprising at least one coding sequence, whereinthe coding sequence comprises or consists of any one of the (modified)nucleic acid sequences defined in SEQ ID NOs: 8821-13230, 39715, 39716,39717, 39720, 39721, 39724, 39725, 39728, 39729, 39730, 39733, 39734,39737, 39738, 39741, 39742, 39745, 39746, and/or SEQ ID NOs:25451-30870, and/or SEQ ID NOs: 30871-35280, and/or SEQ ID NOs:35281-39690, and/or SEQ ID NO: 39713 to SEQ ID NO:39746, and/or SEQ IDNO: 39714, 39716, 39729, 39734, 39738, 39725, or of a fragment orvariant of any one of these sequences. In other words, the at least onecoding sequence preferably comprises or consists of a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 8821-13230,39715, 39716, 39717, 39720, 39721, 39724, 39725, 39728, 39729, 39730,39733, 39734, 39737, 39738, 39741, 39742, 39745, 39746, and/or SEQ IDNOs: 26461-30870, and/or SEQ ID NOs: 30871-35280, and/or SEQ ID NOs:35281-39690, and/or SEQ ID NO: 39713 to SEQ ID NO: 39746, and/or SEQ IDNO: 39714, 39716, 39729, 39734, 39738, 39725, or a fragment or variantof any one of these nucleic acid sequences.

In a further preferred embodiment, the at least one coding sequence ofthe RNA according to the invention comprises or consists of a nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even mare preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (modified) nucleic acid sequencesdefined in SEQ ID NOs: 8821-13230, 39715, 39716, 39717, 39720, 39721,39724, 39725, 39728, 39729, 39730, 39733, 39734, 39737, 39738, 39741,39742, 39745, 39745, and/or SEQ ID NOs: 25451-30870, and/or SEQ ID NOs:30871-35280, and/or SEQ ID NOs: 35281-39690, and/or SEQ ID NO: 39713 toSEQ ID NO: 39746, and/or SEQ ID NOs: 39714, 39716, 39729, 39734, 39738,39725, or of a fragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA according to the invention comprises orconsists of a nucleic acid sequence having a sequence identity of atleast 80% with any one of the (modified) nucleic acid sequences definedin SEQ ID NOs: 8821-13230, 39715, 39716, 39717, 39720, 39721, 39724,39725, 39728, 39729, 39730, 39733, 39734, 39737, 39738, 39741, 39742,39745, 39746, and/or SEQ ID NOs: 25461-30870, and/or SEQ ID Hs:30871-35280, and/or SEQ ID NOs: 35281-39690, and/or SEQ ID NO: 39713 toSEQ ID NO: 39745, and/or SEQ ID NOs: 39714, 39715, 39729, 39734, 39738,39725, or of a fragment or variant of any one of these sequences.

GC Optimized Sequences:

In a preferred embodiment, the present invention provides an RNAcomprising at least one coding sequence, wherein the coding sequencecomprises or consists of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 39714, 39716, 39729, 39734, 39738, 39725, or afragment or variant of any one of these nucleic acid sequences.

According to a further embodiment, the at least one coding sequence ofthe RNA according to the invention comprises or consists of a nucleicacid sequence having a sequence identity of at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, morepreferably of at least 80%, even more preferably at least 85%, even morepreferably of at least 90% and most preferably of at least 95% or even97%, with a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 39714, 39715, 39729, 39734, 39738, 39725, or a fragment orvariant of any one of these nucleic acid sequences.

Sequences Adapted to Human Codon Usage:

According to the invention, a further preferred modification of the RNAof the present invention is based on the finding that codons encodingthe same amino acid typically occur at different frequencies. Accordingto the invention, in the modified RNA of the present invention, thecoding sequence (coding region) as defined herein is preferably modifiedcompared to the corresponding region of the respective wild type RNAsuch that the frequency of the codons encoding the same amino acidcorresponds to the naturally occurring frequency of that codon accordingto the human codon usage as e.g. shown in Table 2.

For example, in the case of the amino acid alanine (Ala) present in anamino acid sequence encoded by the at least one coding sequence of theRNA according to the invention, the wild type coding sequence ispreferably adapted in a way that the codon “GCC” is used with afrequency of 0.40, the codon “GCT” is used with a frequency of 0.28, thecodon “GCA” is used with a frequency of 0.22 and the codon “GCG” is usedwith a frequency of 0.10 etc. (see Table 2).

TABLE 2 Human codon usage table Amino acid codon fraction /1000Amino acid codon fraction /1000 Ala GCG 0.10 7.4 Pro CCG 0.11 6.9 AlaGCA 0.22 15.8 Pro CCA 0.27 16.9 Ala GCT 0.28 18.5 Pro CCT 0.29 17.5 AlaGCC* 0.40 27.7 Pro CCC* 0.33 19.8 Cys TGT 0.42 10.6 Gln CAG* 0.73 34.2Cys TGC* 0.58 12.6 Gln CAA 0.27 12.3 Asp GAT 0.44 21.8 Arg AGG 0.22 12.0Asp GAC* 0.56 25.1 Arg AGA* 0.21 12.1 Glu GAG* 0.59 39.6 Arg CGG 0.1911.4 Glu GAA 0.41 29.0 Arg CGA 0.10 6.2 Phe TTT 0.43 17.6 Arg CGT 0.094.5 Phe TTC* 0.57 20.3 Arg CGC 0.19 10.4 Gly GGG 0.23 16.5 Ser AGT 0.1412.1 Gly GGA 0.26 16.5 Ser AGC* 0.25 19.5 Gly GGT 0.18 10.8 Ser TCG 0.094.4 Gly GGC* 0.33 22.2 Ser TCA 0.15 12.2 His CAT 0.41 10.9 Ser TCT 0.1815.2 His CAC* 0.59 15.1 Ser TCC 0.23 17.7 Ile ATA 0.14 7.5 Thr ACG 0.129.1 Ile ATT 0.35 19.0 Thr ACA 0.27 15.1 Ile ATC* 0.52 20.8 Thr ACT 0.2313.1 Lys AAG* 0.60 31.9 Thr ACC* 0.38 18.9 Lys AAA 0.40 24.4 Val GTG*0.48 28.1 Leu TTG 0.12 12.9 Val GTA 0.10 7.1 Leu TTA 0.09 7.7 Val GTT0.17 11.0 Leu CTG* 0.43 39.9 Val GTC 0.25 14.5 Leu CTA 0.07 7.2 Trp TGG*1 13.2 Leu CTT 0.12 13.2 Tyr TAT 0.42 12.2 Leu CTC 0.20 19.9 Tyr TAC*0.58 15.3 Met ATG* 1 22.0 Stop TGA* 0.91 1.9 Asn AAT 0.44 17.0 Stop TAG0.17 0.8 Asn AAC* 0.59 19.1 Stop TAA 0.22 1.0 *most frequent codon

In a preferred embodiment, the present invention provides an RNAcomprising at least one coding sequence, wherein the coding sequencecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 17641-22050, or a fragment or variant of any one of saidnucleic acid sequences.

According to a further embodiment, the at least one coding sequence ofthe RNA according to the invention comprises or consists of a nucleicacid sequence having a sequence identity of at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 89%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 99%, 97%, 98%, or 99%, preferably of at least 70%, morepreferably of at least 80%, even more preferably at least 85%, even morepreferably of at least 90% and mast preferably of at least 95% or even97%, with a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 17641-22050, or a fragment or variant of any one of saidnucleic acid sequences.

Codon-Optimized Sequences:

As described above it is preferred according to the invention, that allcodons of the wild type sequence which code for a tRNA, which isrelatively rare in the cell, are exchanged for a codon which codes for atRNA, which is relatively frequent in the cell and which, in each case,carries the same amino acid as the relatively rare tRNA. Therefore it isparticularly preferred that the most frequent codons are used for eachencoded amino acid (see Table 2, most frequent codons are marked withasterisks). Such an optimization procedure increases the codonadaptation index (CAI) and ultimately maximises the CAI. In the contextof the invention, sequences with increased or maximized CAI aretypically referred to as “codon-optimized” sequences and/or CAIincreased and/or maximized sequences. According to a preferredembodiment, the RNA of the present invention comprises at least onecoding sequence, wherein the coding sequence is codon-optimized asdescribed herein. More preferably, the codon adaptation index (CAI) ofthe at least one coding sequence is at least 0.5, at least 0.8, at least0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI)of the at least one coding sequence is 1.

For example, in the case of the amino acid alanine (Ala) present in theamino acid sequence encoded by the at least one coding sequence of theRNA according to the invention, the wild type coding sequence is adaptedin a way that the most frequent human codon “GCC” is always used forsaid amino acid, or for the amino acid Cysteine (Cys), the wild typesequence is adapted in a way that the most frequent human codon “TGC” isalways used for said amino acid etc.

In a preferred embodiment, the present invention provides an RNAcomprising at least one coding sequence, wherein the coding sequencecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 22051-29490, or a fragment or variant of any one of saidnucleic acid sequences.

According to a further embodiment, the at least one coding sequence ofthe RNA according to the invention comprises or consists of a nucleicacid sequence having a sequence identity of at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 89%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 98%, 97%, 98%, or 99%, preferably of at least 70%, marepreferably of at least 80%, even more preferably at least 85%, even morepreferably of at least 90% and most preferably of at least 95% or even97%, with a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 22051-29460, or a fragment or variant of any one of saidnucleic acid sequences.

C-Optimized Sequences:

According to another embodiment, the RNA of the composition of thepresent invention may be modified by modifying, preferably increasing,the cytosine (C) content of the RNA, preferably of the coding region ofthe RNA.

In a particularly preferred embodiment of the present invention, the Ccontent of the coding region of the RNA of the present invention ismodified, preferably increased, compared to the C content of the codingregion of the respective wild type RNA, i.e. the unmodified RNA. Theamino acid sequence encoded by the at least one coding sequence of theRNA of the present invention is preferably not modified as compared tothe amino acid sequence encoded by the respective wild type mRNA.

In a preferred embodiment of the present invention, the modified RNA ismodified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, orat least 90% of the theoretically possible maximum cytosine-content oreven a maximum cytosine-content is achieved.

In further preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or even 100% of the codons of the target RNA wild typesequence, which are “cytosine content optimizable” are replaced bycodons having a higher cytosine-content than the ones present in thewild type sequence.

In a further preferred embodiment, some of the codons of the wild typecoding sequence may additionally be modified such that a codon for arelatively rare tRNA in the cell is exchanged by a codon for arelatively frequent tRNA in the cell, provided that the substitutedcodon for a relatively frequent tRNA carries the same amino acid as therelatively rare tRNA of the original wild type codon. Preferably, all ofthe codons for a relatively rare tRNA are replaced by a codon for arelatively frequent tRNA in the cell, except codons encoding aminoacids, which are exclusively encoded by codons not containing anycytosine, or except for glutamine (Gin), which is encoded by two codonseach containing the same number of cytosines.

In a further preferred embodiment of the present invention, the modifiedtarget RNA is modified such that at least 80%, or at least 90% of thetheoretically possible maximum cytosine-content or even a maximumcytosine-content is achieved by means of codons, which code forrelatively frequent tRNAs in the cell, wherein the amino acid sequenceremains unchanged.

Due to the naturally occurring degeneracy of the genetic code, more thanone codon may encode a particular amino acid. Accordingly, 18 out of 20naturally occurring amino acids are encoded by more than one codon (withTryp and Met being an exception), e.g. by 2 codons (e.g. Cys, Asp, Glu),by three codons (e.g. Ile), by 4 codons (e.g. Al, Gly, Pro) or by 6codons (e.g. Leu, Arg, Ser). However, not all codons encoding the sameamino acid are utilized with the same frequency under in vivoconditions. Depending on each single organism, a typical codon usageprofile is established.

The term “cytosine content-optimizable codon” as used within the contextof the present invention refers to codons, which exhibit a lower contentof cytosines than other codons encoding the same amino acid.Accordingly, any wild type codon, which may be replaced by another codonencoding the same amino acid and exhibiting a higher number of cytosineswithin that codon, is considered to be cytosine-optimizable(C-optimizable). Any such substitution of a C-optimizable wild typecodon by the specific C-optimized codon within a wild type coding regionincreases its overall C content and reflects a C-enriched modified mRNAsequence. According to a preferred embodiment, the RNA of the presentinvention, preferably the at least one coding sequence of the RNA of thepresent invention comprises or consists of a C-maximized RNA sequencecontaining C-optimized codons for all potentially C-optimizable codons.Accordingly, 100% or all of the theoretically replaceable C-optimizablecodons are preferably replaced by C-optimized codons over the entirelength of the coding region.

In this context, cytosine-content optimizable codons are codons, whichcontain a lower number of cytosines than other codons coding for thesame amino acid. Any of the codons GCG, GCA, GCU codes for the aminoacid Ala, which may be exchanged by the codon GCC encoding the sameamino acid, and/or the codon HU that codes for Cys may be exchanged bythe codon UGC encoding the same amino acid, and/or the codon GAU whichcodes for Asp may be exchanged by the codon GAC encoding the same aminoacid, and/or the codon that UUU that codes for Phe may be exchanged forthe codon UUC encoding the same amino acid, and/or any of the codonsGGG, GGA, GGU that code Gly may be exchanged by the codon GGC encodingthe same amino acid, and/or the codon CAU that codes for His may beexchanged by the codon CAC encoding the same amino acid, and/or any ofthe codons AUA, AUU that code for Ile may be exchanged by the codon AUC,and/or any of the codons UUG, UUA, CUG, CUA, CUU coding for Leu may beexchanged by the codon CUC encoding the same amino acid, and/or thecodon AAU that codes for Asn may be exchanged by the codon AAC encodingthe same amino acid, and/or any of the codons CCG, CCA, CCU coding forPro may be exchanged by the codon CCG encoding the same amino acid,and/or any of the codons AGG, AGA, CGG, CGA, CGU coding for Arg may beexchanged by the codon CGC encoding the same amino acid, and/or any ofthe codons AGU, AGC, UCG, UCA, UCU coding for Ser may be exchanged bythe codon UCC encoding the same amino acid, and/or any of the codonsACG, ACA, ACU coding for Thr may be exchanged by the codon ACC encodingthe same amino acid, and/or any of the codons GUG, GUA, GUU coding forVal may be exchanged by the codon GUC encoding the same amino acid,and/or the codon UAU coding for Tyr may be exchanged by the codon UACencoding the same amino acid.

In any of the above instances, the number of cytosines is increased by 1per exchanged codon. Exchange of all non C-optimized codons(corresponding to C-optimizable codons) of the coding region results ina C-maximized coding sequence. In the context of the invention, at least70%, preferably at least 80%, more preferably at least 90%, of the nonC-optimized codons within the at least one coding region of the RNAaccording to the invention are replaced by C-optimized codons.

It may be preferred that for some amino acids the percentage ofC-optimizable codons replaced by C-optimized codons is less than 70%,while for other amino acids the percentage of replaced codons is higherthan 70% to meet the overall percentage of C-optimization of at least70% of all C-optimizable wild type codons of the coding region.

Preferably, in a C-optimized RNA of the invention, at least 50% of theC-optimizable wild type codons for any given amino acid are replaced byC-optimized codons, e.g. any modified C-enriched RNA preferably containsat least 50% C-optimized codons at C-optimizable wild type codonpositions encoding any one of the above mentioned amino acids Ala, Cys,Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val and Tyr,preferably at least 00%.

In this context codons encoding amino acids, which are not cytosinecontent-optimizable and which are, however, encoded by at least twocodons, may be used without any further selection process. However, theradon of the wild type sequence that codes for a relatively rare tRNA inthe cell, e.g. a human cell, may be exchanged for a codon that codes fora relatively frequent tRNA in the cell, wherein both code for the sameamino acid. Accordingly, the relatively rare codon GAA coding for Glumay be exchanged by the relative frequent codon GAG coding for the sameamino acid, and/or

the relatively rare codon AAA coding for Lys may be exchanged by therelative frequent codon AAG coding for the same amino acid, and/orthe relatively rare codon CAA coding for Gln may be exchanged for therelative frequent codon CAG encoding the same amino acid.

In this context, the amino acids Met (AUG) and Trp (UGG), which areencoded by only one codon each, remain unchanged. Stop codons are notcytosine-content optimized; however, the relatively rare stop codonsamber, ochre (UAA, UAG) may be exchanged by the relatively frequent stopcodon opal (UGA).

The single substitutions listed above may be used individually as wellas in all possible combinations in order to optimize thecytosine-content of the modified RNA compared to the wild type mRNAsequence.

Accordingly, the at least one coding sequence as defined herein may bechanged compared to the coding region of the respective wild type RNA insuch a way that an amino acid encoded by at least two or more codons, ofwhich one comprises one additional cytosine, such a codon may beexchanged by the C-optimized codon comprising one additional cytosine,wherein the amino acid is preferably unaltered compared to the wild typesequence.

In a preferred embodiment, the present invention provides an RNAcomprising at least one coding sequence, wherein the coding sequencecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID Hs: 13231-17640, or a fragment or variant of any one of saidnucleic acid sequences.

According to a further embodiment, the at least one coding sequence ofthe RNA according to the invention comprises or consists of a nucleicacid sequence having a sequence identity of at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 89%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, morepreferably of at least 80%, even more preferably at least 85%, even morepreferably of at least 90% and most preferably of at least 95% or even97%, with a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 13231-17940, or a fragment or variant of any one of saidnucleic acid sequences.

According to a particularly preferred embodiment, the invention providesan RNA, preferably an mRNA, comprising at least one coding sequence asdefined herein, wherein the G/C content of the at least one codingsequence of the RNA is increased compared to the G/C content of thecorresponding coding sequence of the corresponding wild type RNA, and/orwherein the C content of the at least one coding sequence of the RNA isincreased compared to the C content of the corresponding coding sequenceof the corresponding wild type RNA, and/or wherein the codons in the atleast one coding sequence of the RNA are adapted to human codon usage,wherein the codon adaptation index (CAI) is preferably increased ormaximised in the at least one coding sequence of the RNA, and whereinthe amino acid sequence encoded by the RNA is preferably not beingmodified compared to the amino acid sequence encoded by thecorresponding wild type RNA.

5′-Cap Structure:

Modification of the 5′-End of a Modified Artificial Nucleic Acid:

According to another preferred embodiment of the invention, theartificial nucleic acid, preferably an mRNA, as defined herein, can bemodified by the addition of a so-called “5′-cap” structure, whichpreferably stabilizes the nucleic acid, preferably an mRNA, as describedherein.

In a particularly preferred embodiment, the artificial nucleic acidaccording to the invention, preferably an mRNA, comprises a 5′-capstructure.

A 5′-cap is an entity, typically a modified nucleotide entity, whichgenerally “caps” the 5′-end of a nucleic acid, for example of a maturemRNA. A 5′-cap may typically be formed by a modified nucleotide,particularly by a derivative of a guanine nucleotide. Preferably, the5′-cap is linked to the 5′-terminus via a 5¹-5′-triphosphate linkage. A5′-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5′nucleotide of the nucleic acid carrying the 5′-cap, typically the 5′-endof an mRNA. m7GpppN is the 5′-cap structure, which naturally occurs inmRNA transcribed by polymerase II and is therefore preferably notconsidered as modification comprised in an artificial nucleic acid inthis context. Accordingly, a modified artificial nucleic acid,preferably an mRNA, of the present invention may comprise an m7GpppN as5′-cap, but additionally the modified artificial nucleic acid,preferably an mRNA, typically comprises at least one furthermodification as defined herein.

Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-cap structuresare regarded as at least one modification in this context.

Particularly preferred modified 5′-cap structures are cap1 (methylationof the ribose of the adjacent nucleotide of m70), cap2 (additionalmethylation of the ribose of the 2nd nucleotide downstream of the m70),cap3 (additional methylation of the ribose of the 3rd nucleotidedownstream of the m7G), cap4 (additional methylation of the ribose ofthe 4th nucleotide downstream of the m70), ARCA (anti-reverse capanalogue, modified ARCA (e.g. phosphothioate modified ARCA), inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine.

5′-UTRs:

According to a further embodiment, the artificial nucleic acid comprisesan untranslated region (UTR). More preferably, the artificial nucleicacid according to the invention, preferably an mRNA, comprises at leastone of the following structural elements: a 5′- and/or 3′-untranslatedregion element (UTR element), particularly a 5′-UTR element, whichcomprises or consists of a nucleic acid sequence which is derived fromthe 5′-UTR of a TOP gene or from a fragment, homolog or a variantthereof, or a 5′- and/or 3′-UTR element which may be derivable from agene that provides a stable mRNA or from a homolog, fragment or variantthereof; a histone-stem-loop structure, preferably a histone-stem-loopin its 3′ untranslated region; a 5′-cap structure; a poly-A tail; or apoly(C) sequence.

In a preferred embodiment, the artificial nucleic acid, preferably anmRNA, comprises at least one 5′- or 3′-UTR element. In this context, anUTR element comprises or consists of a nucleic acid sequence, which isderived from the 5′- or 3′-UTR of any naturally occurring gene or whichis derived from a fragment, a homolog or a variant of the 5′- or 3′-UTRof a gene. Preferably the 5′- or 3′-UTR element used according to thepresent invention is heterologous to the coding region of the inventiveartificial nucleic acid. Even if 5′- or 3′-UTR elements derived fromnaturally occurring genes are preferred, also synthetically engineeredUTR elements may be used in the context of the present invention.

According to a preferred embodiment, the artificial nucleic acidaccording to the invention comprises a 5′-UTR. More preferably, theartificial nucleic acid comprises a 5′-UTR comprising at least oneheterologous 5′-UTR element.

In a particularly preferred embodiment, the artificial nucleic acidcomprises at least one 5′-untranslated region element (5′-UTR element),preferably a heterologous 5′-UTR element, which comprises or consists ofa nucleic acid sequence, which is derived from the 5′-UTR of a TOP geneor which is derived from a fragment, homolog or variant of the 5′-UTR ofa TOP gene.

It is particularly preferred that the 5′-UTR element does not comprise aTOP-motif or a 5′TOP, as defined above.

In same embodiments, the nucleic acid sequence of the 5′-UTR element,which is derived from a 5′-UTR of a TOP gene, terminates at its 3′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it isderived from. Thus, the 5′-UTR element does not comprise any part of theprotein coding region. Thus, preferably, the only protein coding part ofthe artificial nucleic acid is provided by the at least one codingregion.

The nucleic acid sequence, which is derived from the 5′-UTR of a TOPgene, is typically derived from a eukaryotic TOP gene, preferably aplant or animal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′-UTR element is preferably selected from 5′-UTRelements comprising or consisting of a nucleic acid sequence, which isderived from a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 1-1363, SEQ ID NO:1395, SEQ ID NO: 1421 and SEQ ID NO:1422 of the patent application WO 2013/143700, whose disclosure isincorporated herein by reference, from the homologs of SEQ ID NOs:1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of thepatent application WO 2013/143700, from a variant thereof, or preferablyfrom a corresponding RNA sequence. The term “homologs of SEQ ID Hs:1-1393, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of thepatent application WO 2013/143700” refers to sequences of other speciesthan homo sapiens, which are homologous to the sequences according toSEQ ID NOs: 1-1393, SEQ ID NO:1395, SEQ ID NO: 1421 and SEQ ID NO: 1422of the patent application WO 2013/143700.

In a preferred embodiment, the 5′-UTR element of the artificial nucleicacid, preferably an mRNA, comprises or consists of a nucleic acidsequence, which is derived from a nucleic acid sequence extending fromnucleotide position 5 (i.e. the nucleotide that is located at position 5in the sequence) to the nucleotide position immediately 5′ to the startcodon (located at the 3′ end of the sequences), e.g. the nucleotideposition immediately 5′ to the ATG sequence, of a nucleic acid sequenceselected from SEQ ID NOs: 1-1393, SEQ ID NO:1395, SEQ ID NO:1421 and SEQID NO: 1422 of the patent application WO 2013/143700, from the homologsof SEQ ID NOs: 1-1393, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of the patent application WO 2013/143700 from a variant thereof, ora corresponding RNA sequence. It is particularly preferred that the5′-UTR element is derived from a nucleic acid sequence extending fromthe nucleotide position immediately 3′ to the 5′TOP to the nucleotideposition immediately 5′ to the start codon (located at the 3′-end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID NOs: 1-1393,SEQ ID NO:1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patentapplication WO 2013/143700, from the homologs of SEQ ID NOs: 1-1393, SEQID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patentapplication WO 2013/143700, from a variant thereof, or a correspondingRNA sequence.

In a particularly preferred embodiment, the 5′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′-UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′-UTR of aTOP gene encoding a ribosomal protein. For example, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 5′-UTR of a nucleic acid sequence according to any of SEQ ID NO: 97,170, 193, 244, 259, 554, 950, 975, 700, 721, 913, 1016, 1093, 1120,1138, and 1284-1390 of the patent application WO 2013/143700, acorresponding RNA sequence, a homing thereof, or a variant thereof asdescribed herein, preferably lacking the 5′TOP motif. As describedabove, the sequence extending from position 5 to the nucleotideimmediately 5′ to the ATG (which is located at the 3′-end of thesequences) corresponds to the 5′-UTR of said sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 1-4410, or a fragment or variant of any ofthese sequences, wherein these sequences resemble VP1 protein sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 4411-8820, 39713, 39714, 39718, 39719,39722, 39723, 39726, 39727, 39731, 39732, 39735, 39736, 39739, 39740,39743 and 39744, or a fragment or variant of any of these sequences,wherein these sequences resemble VP1 nucleotide wild type sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 8821-13230, 39715, 39719, 39717, 39720,39721, 39724, 39725, 39728, 39729, 39730, 39733, 39734, 39737, 39738,39741, 39742, 39745, 39746, and/or SEQ ID NOs: 26461-30870, and/or SEQID NOs: 30871-35280, and/or SEQ ID NOs: 35781-39690, and/or SEQ IDNO:39713 to SEQ ID NO:39746, and/or SEQ ID NO: 39714, 39716, 39729,39734.39738, 39725, or a fragment or variant of any of these sequences,wherein these sequences resemble optimized VP1 nucleotide wild typesequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 13231-17640, or a fragment or variant of anyof these sequences, wherein these sequences resemble optimized VP1nucleotide wild type sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 17641-22050, or a fragment or variant of anyof these sequences, wherein these sequences resemble optimized VP1nucleotide wild type sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 22051-26460, or a fragment or variant of anyof these sequences, wherein these sequences resemble optimized VP1nucleotide wild type sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 26461-30870, or a fragment or variant of anyof these sequences, wherein these sequences resemble optimized VP1nucleotide wild type sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 30871-35280, or a fragment or variant of anyof these sequences, wherein these sequences resemble optimized VP1nucleotide wild type sequences.

In one embodiment, the sequences of the invention are selected from thegroup of any of SEQ ID NOs: 35281-39690, or a fragment or variant of anyof these sequences, wherein these sequences resemble optimized VP1nucleotide wild type sequences.

Preferably, the artificial nucleic acid according to the inventioncomprises a 5′-UTR comprising at least one heterologous 5′-UTR sequence,wherein the at least one heterologous 5′-UTR element comprises a nucleicacid sequence, which is derived from a 5′-UTR of a TOP gene encoding aribosomal protein, preferably from a corresponding RNA sequence, or froma homolog, a fragment or a variant thereof, preferably lacking the 5′TOPmotif.

Preferably, the 5′-UTR element comprises or consists of a nucleic acidsequence, which is derived from a 5′-UTR of a TOP gene encoding aribosomal Large protein (RPL) or from a homolog or variant of a 5′-UTRof a TOP gene encoding a ribosomal Large protein (RPL). For example, the5′-UTR element comprises or consists of a nucleic acid sequence, whichis derived from a 5′-UTR of a nucleic acid sequence according to any ofSEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421and 1422 of the patent application WO 2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′TOP motif.

In a particularly preferred embodiment, the 5′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from the 5′-UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, or from a variant of the 5′-UTRof a ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, wherein preferably the 5′-UTRelement does not comprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NO: 20649 (5′-UTRof human ribosomal protein Large 32 lacking the 5′-terminaloligopyrimidine tract; corresponding to SEQ ID NO: 1368 of the patentapplication WO 2013/143700) or preferably to a corresponding RNAsequence, such as SEQ ID NO: 39692, or wherein the at least one 5′-UTRelement comprises or consists of a fragment of a nucleic acid sequencewhich has an identity of at least about 40%, preferably of at leastabout 50%, preferably of at least about 60%, preferably of at leastabout 70%, more preferably of at least about 80%, more preferably of atleast about 90%, even more preferably of at least about 95%, even morepreferably of at least about 99% to the nucleic acid sequence accordingto SEQ ID NO: 39691, or more preferably to a corresponding RNA sequence,such as SEQ ID NO: 39692, wherein, preferably, the fragment is asdescribed above, i.e. being a continuous stretch of nucleotidesrepresenting at least 20% etc. of the full-length 5′-UTR. Preferably,the fragment exhibits a length of at least about 20 nucleotides or more,preferably of at least about 30 nucleotides or more, more preferably ofat least about 40 nucleotides or more. Preferably, the fragment is afunctional fragment as described herein.

In some embodiments, the artificial nucleic acid according to theinvention comprises a 5′-UTR element, which comprises or consists of anucleic acid sequence, which is derived from the 5′-UTR of a vertebrateTOP gene, such as a mammalian, e.g. a human TOP gene, selected fromRPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10,RPS11, RPS12, RPS13, RPS14, RPS5, RPS15A, RPS16, RPS17, RPS18, RPS19,RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RP327, RPS27A, RPS28, RPS29,RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A,RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19,RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29,RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A,RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1,RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3,EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52,NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or from a homolog or variant thereof,wherein preferably the 5′-UTR element does not comprise a TOP-motif orthe STOP of said genes, and wherein optionally the 5′-UTR element startsat its 5′-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 downstream of the 5′-terminal oligopyrimidine tract (TOP) andwherein further optionally the 5′-UTR element which is derived from a5′-UTR of a TOP gene terminates at its 3′-end with a nucleotide locatedat position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon(A(U/T)G) of the gene it is derived from.

According to a preferred embodiment, the artificial nucleic acidcomprises at least one heterologous 5′-UTR element comprising a nucleicacid sequence, which is derived from a 5′-UTR of a TOP gene encoding aribosomal Large protein (RPL), preferably RPL32 or RPL35A, or from agene selected from the group consisting of HSD17134, ATP5A1, AIG1,ASAH1, COX6C or ABCB7 (also referred to herein as MDR), or from ahomolog, a fragment or variant of any one of these genes, preferablylacking the 5′TOP motif.

In further particularly preferred embodiments, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which is derived fromthe 5′-UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomalprotein Large 35 gene (RPL35), a ribosomal protein Large 21 gene(RPL21), an ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, an hydroxysteroid(17-beta) dehydrogenase 4 gene (HSD17B4), an androgen-induced 1 gene(AIG1), cytochrome c oxidase subunit Vlc gene (COX6C), aN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1), or anATP-Binding Cassette, Sub-Family B (MDR/TAP), Member 7 gene (ABCB7), orfrom a variant thereof, preferably from a vertebrate ribosomal proteinLarge 32 gene (RPL32), a vertebrate ribosomal protein Large 35 gene(RPL35), a vertebrate ribosomal protein Large 21 gene (RPL21), avertebrate ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, a vertebratehydroxysteroid (17-beta) dehydrogenase 4 gene (HSD1784), a vertebrateandrogen-induced 1 gene (AIG1), a vertebrate cytochrome c oxidasesubunit VIc gene (COX6C), a vertebrate N-acylsphingosine amidohydrolase(acid ceramidase) 1 gene (ASAH1), or a vertebrate ATP-Binding Cassette,Sub-Family B (MDR/TAP), Member 7 gene (ABCB7), or from a variantthereof, mare preferably from a mammalian ribosomal protein Large 32gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomalprotein Large 21 gene (RPL21), a mammalian ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle(ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammaliancyto-chrome c oxidase subunit VIc gene (COX6C), a mammalianN-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1), or amammalian ATP-Binding Cassette, Sub-Family B (MDR/TAP), Member 7 gene(ABCB7), or from a variant thereof, most preferably from a humanribosomal protein Large 32 gene (RPL32), a human ribosomal protein Large35 gene (RPL35), a human ribosomal protein Large 21 gene (RPL21), ahuman ATP synthase, H+ transporting, mitochondrial F1 complex, alphasubunit 1, cardiac muscle (ATP5A1) gene, a human hydroxysteroid(17-beta) dehydrogenase 4 gene (HS017B4), a human androgen-induced 1gene (AIG1), a human cytochrome c oxidase subunit VIc gene (COX6C), ahuman N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1),or a human ATP-Binding Cassette, Sub-Family B (MDR/TAP), Member 7 gene(ABCB7), or from a variant thereof, wherein preferably the 5′-UTRelement does not comprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NO: 1368, or SEQ IDNOs: 1412-1420 of the patent application WO 2013/143700, or acorresponding RNA sequence, or wherein the at least one 5′-UTR elementcomprises or consists of a fragment of a nucleic acid sequence which hasan identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID NO:1368, or SEQ ID NOs: 1412-1420 of the patent application WO 2013/143700,wherein, preferably, the fragment is as described above, i.e. being acontinuous stretch of nucleotides representing at least 20% etc. of thefull-length 5′-UTR. Preferably, the fragment exhibits a length of atleast about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

According to a particularly preferred embodiment, the artificial nucleicacid comprises a 5′-UTR comprising at least one heterologous 5′-UTRelement, wherein the heterologous 5′-UTR element comprises a nucleicacid sequence according to SEQ ID NO: 39691 to SEQ ID NO: 39694, or ahomolog, a fragment or a variant thereof. Preferably, the at least oneheterologous 5′-UTR element comprises or consists of a nucleic acidsequence, which has an identity of at least about 40%, preferably of atleast about 50%, preferably of at least about 60%, preferably of atleast about 70%, more preferably of at least about 80%, more preferablyof at least about 90%, even more preferably of at least about 95%, evenmore preferably of at least about 99% to a nucleic acid sequenceaccording to any one of SEQ ID NO: 39691 to SEQ ID NO: 39694.

According to a preferred embodiment, the artificial nucleic acidaccording to the invention comprises a 3′-untranslated region (3′-UTR).More preferably, the artificial nucleic acid according to the inventioncomprises a 3′-UTR comprising or consisting of at least one heterologous3′-UTR element, preferably as defined herein.

Poly(A) Sequence and Poly(C) Sequence:

According to a further preferred embodiment, the artificial nucleicacid, preferably the 3′-UTR, may contain a poly-A tail of typicallyabout 10 to 200 adenosine nucleotides, preferably about 10 to 100adenosine nucleotides, more preferably about 40 to 80 adenosinenucleotides or even more preferably about 50 to 70 adenosinenucleotides.

Preferably, the poly(A) sequence in the artificial nucleic acid,preferably an mRNA, is derived from a DNA template by in vitrotranscription. Alternatively, the poly(A) sequence may also be obtainedin vitro by common methods of chemical-synthesis without beingnecessarily transcribed from a DNA progenitor. Moreover, poly(A)sequences, or poly(A) tails may be generated by enzymaticpolyadenylation of the RNA according to the present invention usingcommercially available polyadenylation kits and corresponding protocolsknown in the art, or using immobilized poly(A)polymerases e.g. in apolyadenylation reactor (WO 2016/174271).

Alternatively, the artificial nucleic acid, preferably an mRNA,optionally comprises a polyadenylation signal, which is defined hereinas a signal, which conveys polyadenylation to a (transcribed) mRNA byspecific protein factors (e.g. cleavage and polyadenylation specificityfactor (CPSF), cleavage stimulation factor (CstF), cleavage factors Iand II (CF I and CF II), poly(A) polymerase (PAP)). In this context, aconsensus polyadenylation signal is preferred comprising the NN(U/T)ANAconsensus sequence. In a particularly preferred aspect, thepolyadenylation signal comprises one of the following sequences:AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usually present in RNAand thymidine is usually present in DNA).

According to a further preferred embodiment, the artificial nucleic acidof the present invention, preferably the 3′-UTR of the artificialnucleic acid, may contain a poly-C tail of typically about 10 to 200cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides,more preferably about 20 to 70 cytosine nucleotides or even morepreferably about 20 to 60 or even 10 to 40 cytosine nucleotides.

3′-UTRs:

In a further preferred embodiment, the artificial nucleic acid accordingto the invention further comprises at least one 3′-UTR element, whichcomprises or consists of a nucleic acid sequence derived from the 3′-UTRof a chordate gene, preferably a 10 vertebrate gene, more preferably amammalian gene, most preferably a human gene, or from a variant of the3′-UTR of a chordate gene, preferably a vertebrate gene, more preferablya mammalian gene, most preferably a human gene.

The term “3′-UTR element” refers to a nucleic acid sequence, whichcomprises or consists of a nucleic acid sequence that is derived from a3′-UTR or from a variant of a T-UTR. A 3′-UTR element in the sense ofthe present invention may represent the 3′-UTR on a DNA or on an RNAlevel. Thus, in the sense of the present invention, preferably, a 3′-UTRelement may be the 3′-UTR of an mRNA, preferably of an artificial mRNA,or it may be the transcription template for a 3′-UTR of an mRNA. Thus, a3′-UTR element preferably is a nucleic acid sequence, which correspondsto the 3′-UTR of an mRNA, preferably to the 3′-UTR of an artificialmRNA, such as an mRNA obtained by transcription of a geneticallyengineered vector construct. Preferably, the 3′-UTR element fulfils thefunction of a 3′-UTR or encodes a sequence, which fulfils the functionof a 3′-UTR.

Preferably, the artificial nucleic acid comprises a 3′-UTR elementcomprising or consisting of a nucleic acid sequence derived from a3′-UTR of a gene, which preferably encodes a stable mRNA, or from ahomolog, a fragment or a variant of said gene. In particular, the 3′-UTRelement may be derivable from a gene that relates to an mRNA with anenhanced half-life (that provides a stable mRNA), for example a 3′-UTRelement as defined and described below.

In a particularly preferred embodiment, the 3′-UTR element comprises orconsists of a nucleic acid sequence which is derived from a 3′-UTR of agene selected from the group consisting of an albumin gene, an α-globingene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene,and a collagen alpha gene, such as a collagen alpha 1(I) gene, or from ahomolog, a fragment or a variant of a 3′-UTR of a gene selected from thegroup consisting of an albumin gene, an α-globin gene, a β-globin gene,a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alphagene, such as a collagen alpha 1(I) gene. More preferably, the 3′-UTRelement comprises or consists of a nucleic acid sequence which isderived from a 3′-UTR of a gene selected from the group consisting of analbumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylasegene, a lipoxygenase gene, and a collagen alpha gene, such as a collagenalpha 1(I) gene, or from a homolog, a fragment or a variant of a 3′-UTRof a gene selected from the group consisting of an albumin gene, anα-globin gene, a β-globin gene, a tyrosine hydroxylase gene, alipoxygenase gene, and a collagen alpha gene, such as a collagen alpha1(I) gene according to SEQ ID NOs: 1369-1390 of the patent applicationWO 2013/143700, whose disclosure is incorporated herein by reference, orfrom a homolog, a fragment or a variant thereof.

In a particularly preferred embodiment, the 3′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from the 3′-UTR ofa vertebrate albumin gene or from a variant thereof, preferably from the3′-UTR of a mammalian albumin gene or from a variant thereof, morepreferably from the 3′-UTR of a human albumin gene or from a variantthereof, even more preferably from the 3′-UTR of the human albumin geneaccording to Genbank Accession number NM_000477.5, or from a fragment orvariant thereof. More preferably, the 3′-UTR element comprises orconsists of a nucleic acid according to SEQ ID NO: 39703, or SEQ ID NO:39704 (corresponding to SEQ ID NO:1399 of the patent application WO2013/143700), or a fragment, homolog or variant thereof.

Most preferably the 3′-UTR element comprises or consists of the nucleicacid sequence derived from a fragment of the human albumin geneaccording to SEQ ID NO: 39705, or SEQ ID NO: 39709 (corresponding to SEQID NO: 1376 of the patent application WO 2013/143700), or a fragment,homolog or variant thereof. Further preferably, the 3′-UTR elementcomprises or consists of a nucleic acid according to SEQ ID NO: 39707,or SEQ ID NO: 39708, (Albumin 7), or a fragment, homolog or variantthereof.

In another particularly preferred embodiment, the at least oneheterologous 3′-UTR element comprises or consists of a nucleic acidsequence derived from a 3′-UTR of an α-globin gene, preferably avertebrate α- or β-globin gene, more preferably a mammalian α- orβ-globin gene, most preferably a human α- or β-globin gene.

More preferably, the 3′-UTR element comprises or consists of a nucleicacid according to SEQ ID NO: 39695, or SEQ ID NO: 39696 (correspondingto SEQ ID NO: 1370 of the patent application WO 2013/143700), or ahomolog, a fragment, or a variant thereof

Preferably, the at least one heterologous 3′-UTR element comprises orconsists of a nucleic acid sequence derived from a 3′-UTR of Homosapiens hemoglobin, alpha 1 (HBA1). More preferably, the 3′-UTR elementcomprises or consists of a nucleic acid according to SEQ ID NO: 39695,or SEQ ID NO: 39696 (corresponding to SEQ ID NO: 1370 of the patentapplication WD 2013/143700), or a homolog, a fragment, or a variantthereof.

In another embodiment, the at least one heterologous 3′-UTR elementcomprises or consists of a nucleic acid sequence derived from a 3′-UTRof Homo sapiens hemoglobin, alpha 2 (HBA2). More preferably, the 3′-UTRelement comprises or consists of a nucleic acid according to SEQ ID NO:39697 or SEQ ID NO: 39698 (corresponding to SEQ ID NO: 1371 of thepatent application WD 2013/143700), or a homolog, a fragment, or avariant thereof.

According to another embodiment, the at least one heterologous 3′-UTRelement comprises or consists of a nucleic acid sequence derived from a3′-UTR of Homo sapiens hemoglobin, beta (HBB). More preferably, the3′-UTR element comprises or consists of a nucleic acid according to SEQID NO: 39699, or SEQ ID NO: 39700 (corresponding to SEQ ID NO:1372 ofthe patent application WO 2013/143700), or a homolog, a fragment, or avariant thereof.

The at least one heterologous 3′-UTR element may further comprise orconsist of the center, α-complex-binding portion of the 3′-UTR of anα-globin gene, such as of a human α-globin gene, or a homolog, afragment, or a variant of an α-globin gene, preferably according to SEQID NO: 39701 or SEQ ID NO: 39702 (also referred to herein as “muag”)(corresponding to SEQ ID NO: 1393 of the patent application WO2013/143700), or a humping, a fragment, or a variant thereof.

The term “a nucleic acid sequence which is derived from the 3′-UTR of a[ . . . ] gene” preferably refers to a nucleic acid sequence which isbased on the 3′-UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′-UTR of an albumin gene, an α-globin gene, a (β-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, preferably of an albumingene or on a part thereof. This term includes sequences corresponding tothe entire 3′-UTR sequence, i.e. the full length 3′-UTR sequence of agene, and sequences corresponding to a fragment of the 3′-UTR sequenceof a gene, such as an albumin gene, α-globin gene, β-globin gene,tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene,such as a collagen alpha 1(I) gene, preferably of an albumin gene.

The term “a nucleic acid sequence which is derived from a variant of the3′-UTR of a [ . . . ] gene” preferably refers to a nucleic acidsequence, which is based on a variant of the 3′-UTR sequence of a gene,such as on a variant of the 3′-UTR of an albumin gene, an α-globin gene,a (β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or acollagen alpha gene, such as a collagen alpha 1(I) gene, or on a partthereof as described above. This term includes sequences correspondingto the entire sequence of the variant of the 3′-UTR of a gene, i.e. thefull length variant 3′-UTR sequence of a gene, and sequencescorresponding to a fragment of the variant 3′-UTR sequence of a gene. Afragment in this context preferably consists of a continuous stretch ofnucleotides corresponding to a continuous stretch of nucleotides in thefull-length variant 3′-UTR, which represents at least 20%, preferably atleast 30%, more preferably at least 40%, more preferably at least 50%,even more preferably at least 60%, even mare preferably at least 70%,even more preferably at least 80%, and most preferably at least 90% ofthe full-length variant 3′-UTR. Such a fragment of a variant, in thesense of the present invention, is preferably a functional fragment of avariant as described herein.

Preferably, the at least one 5′-UTR element and the at least one 3′-UTRelement act synergistically to increase protein production from theinventive artificial nucleic acid as described above.

Histone-Stem-Loop:

In a particularly preferred embodiment, the inventive artificial nucleicacid as described herein comprises a histone stem-loopsequence/structure (histone stem-loop). Such histone stem-loop sequencesare preferably selected from histone stem-loop sequences as disclosed inWO 2012/019780, whose disclosure is incorporated herewith by reference.

A histone stem-loop sequence, suitable to be used within the presentinvention, is preferably selected from at least one of the followingformulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

-   stem1 or stem2 bordering elements N₁₋₆ is a consecutive sequence of    1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more    preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each    N is independently from another selected from a nucleotide selected    from A, U, T, G and C, or a nucleotide analogue thereof;-   stem1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse    complementary with element stem2, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof, and    -   wherein G is guanosine or an analogue thereof, and may be        optionally replaced by a cytidine or an analogue thereof,        provided that its complementary nucleotide cytidine in stem2 is        replaced by guanosine;-   loop sequence [N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and    stem2, and is a consecutive sequence of 3 to 5 nucleotides, more    preferably of 4 nucleotides;    -   wherein each N₀₋₄ is independent from another a consecutive        sequence of 0 to 4, preferably of 1 to 3, more preferably of 1        to 2 N, wherein each N is independently from another selected        from a nucleotide selected from A, U, T, G and C or a nucleotide        analogue thereof; and    -   wherein U/T represents uridine, or optionally thymidine;-   stem2 [N₃₋₅CN₀₋₂] is reverse complementary or partially reverse    complementary with element stem1, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        or C or a nucleotide analogue thereof; and    -   wherein C is cytidine or an analogue thereof, and may be        optionally replaced by a guanosine or an analogue thereof        provided that its complementary nucleoside guanosine in stem1 is        replaced by cytidine;        wherein        stem1 and stem2 are capable of base pairing with each other        forming a reverse complementary sequence, wherein base pairing        may occur between stem1 and stem2, e.g. by Watson-Crick base        pairing of nucleotides A and U/T or G and C or by        non-Watson-Crick base pairing e.g. wobble base pairing, reverse        Watson-Crick base pairing, Hoogsteen base pairing, reverse        Hoogsteen base pairing or are capable of base pairing with each        other forming a partially reverse complementary sequence,        wherein an incomplete base pairing may occur between stem1 and        stem2, on the basis that one ore more bases in one stem do not        have a complementary base in the reverse complementary sequence        of the other stem.

According to a further preferred embodiment of the first inventiveaspect, the inventive artificial nucleic acid may comprise at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein: N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the inventive artificial nucleic acid may comprise atleast one histone stem-loop sequence according to at least one of thefollowing specific formulae (Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein: N, C, G, T and U are as defined above.

A particular preferred histone stem-loop sequence is the nucleic acidsequence according to SEQ ID NO: 39709, or more preferably thecorresponding RNA sequence according to SEQ ID NO: 39710.

Additional Peptide or Protein Elements:

According to other preferred embodiments, the artificial nucleic acidsequence, particularly the RNA sequence according to the invention mayadditionally encode further peptide or protein elements that e.g.,promote secretion of the protein (secretory signal peptides), promoteanchoring of the encoded antigen in the plasma membrane (transmembranedomains), promote virus-like particle formation (VLP forming domains).In addition, the artificial nucleic acid sequence according to thepresent invention may additionally encode peptide linker elements,self-cleaving peptides or helper peptides.

According to another particularly preferred embodiment, the inventiveartificial nucleic acid may additionally or alternatively encode asecretory signal peptide (signal sequence). Such signal peptides aresequences, which typically exhibit a length of about 10 to 30 aminoacids and are preferably located at the N-terminus of the encodedpeptide, without being limited thereto. Signal peptides as definedherein preferably allow the transport of the at least one proteinencoded by the at least one coding region of the inventive artificialnucleic acid into a defined cellular compartment, preferably the cellsurface, the endoplasmic reticulum (ER) or the endosomal-lysosomalcompartment. Examples of secretory signal peptide sequences as definedherein include, without being limited thereto, signal sequences ofclassical or non-classical MHC-molecules (e.g. signal sequences of MHC Iand II molecules, e.g. of the MHC class I molecule HLA-A*0201), signalsequences of cytokines or immunoglobulines as defined herein, signalsequences of the invariant chain of immunoglobulines or antibodies asdefined herein, signal sequences of Lamp1, Tapasin, Erp57, Calretikulin,Calnexin, and further membrane associated proteins or of proteinsassociated with the endoplasmic reticulum (ER) or theendosomal-lysosomal compartiment. More preferably, signal sequences ofMHC class 1 molecule HLA-A*0201 may be used according to the presentinvention.

According to other embodiments, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one transmembrane domain element.

Transmembrane elements or membrane spanning polypeptide elements arepresent in proteins that are integrated or anchored in plasma membranesof cells. Typical transmembrane elements are alpha-helical transmembraneelements. Such transmembrane elements are composed essentially of aminoacids with hydrophobic side chains, because the interior of a cellmembrane (lipid bilayer) is also hydrophobic. From the structuralperspective, transmembrane elements are commonly single hydrophobicalpha helices or beta barrel structures; whereas hydrophobic alphahelices are usually present in proteins that are present in membraneanchored proteins (e.g., seven transmembrane domain receptors),beta-barrel structures are often present in proteins that generate poresor channels.

For target proteins, such as antigenic peptides or proteins according tothe present invention (derived from Norovirus) it may be beneficial tointroduce a transmembrane element into the respective constructs. Byaddition of a transmembrane element to the target peptide/protein it maybe possible to further enhance the immune response, wherein thetranslated target peptide/protein, e.g. a viral antigen, anchors to atarget membrane, e.g. the plasma membrane of a cell, thereby increasingimmune responses. This effect is also referred to as antigen clustering.

When used in combination with a polypeptide or protein of interest inthe context of the present invention, such transmembrane element can beplaced N-terminal or C-terminal to the Norovirus antigenic peptide orprotein of interest. On nucleic acid level, the coding sequence for suchtransmembrane element is typically placed in frame (i.e. in the samereading frame), 5′ or 3′ to the coding sequence of the polypeptide asdefined herein.

The transmembrane domain may be selected from the transmembrane domainof Hemagglutinin (HA) of Influenza virus, Env of HIV-1, EIAV (equineinfectious anaemia virus), MLV (murine leukaemia virus), mouse mammarytumor virus, G protein of VSV (vesicular stomatitis virus), Rabiesvirus, or a transmembrane element of a seven transmembrane domainreceptor.

According to other embodiments, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one VLP forming domain.

VLPs are self-assembled viral structural proteins (envelope proteins orcapsid proteins) that structurally resemble viruses (without containingviral genetic material). VLPs contain repetitive high density displaysof antigens which present conformational epitopes that can elicit strongT cell and B cell immune responses.

When used in combination with a Norovirus antigenic peptide or proteinin the context of the present invention, such VLP forming element can beplaced N-terminal or C-terminal to the polypeptide of interest. Onnucleic acid level, the coding sequence for such VLP forming element istypically placed in frame (i.e. in the same reading frame), 5′ or 3′ tothe coding sequence of the polypeptide as defined herein.

For nucleic acid (e.g. RNA) encoding a polypeptide or protein ofinterest, particularly Norovirus antigenic polypeptides or proteins, itmay be beneficial to introduce a VLP forming element into the respectiveconstructs. In addition to the “clustering” of epitopes, an improvedsecretion of the VLP particle may also increase the immunogenicity ofthe respective antigen.

VLP forming elements fused to an antigen may generate virus likeparticles containing repetitive high density displays of antigens.Essentially, such VLP forming elements can be chosen from any viral orphage capsid or envelope protein.

According to another embodiment, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one peptide linker element.

In protein constructs composed of several elements (e.g., Norovirusantigenic peptide or protein fused to a transmembrane domain), theprotein elements may be separated by peptide linker elements. Suchelements may be beneficial because they allow for a proper folding ofthe individual elements and thereby the proper functionality of eachelement. Alternatively, the term “spacer” or “peptide spacer” is usedherein.

When used in the context of the present invention, such linkers orspacers are particularly useful when encoded by a nucleic acid encodingat least two functional protein elements, such as at least onepolypeptide or protein of interest (Norovirus antigens) and at least onefurther protein or polypeptide element (e.g., VLP forming domain,transmembrane domain). In that case, the linker is typically located onthe polypeptide chain in between the polypeptide of interest and the atleast one further protein element. On nucleic acid level, the codingsequence for such linker is typically placed in the reading frame, 5′ or3′ to the coding sequence for the polypeptide or protein of interest, orplaced between coding regions for individual polypeptide domains of agiven protein of interest.

Peptide linkers are preferably composed of small, non-polar (e.g. Gly)or polar (e.g. Ser or Thr) amino acids. The small size of these aminoacids provides flexibility, and allows for mobility of the connectingfunctional domains. The incorporation of Ser or Thr can maintain thestability of the linker in aqueous solutions by forming hydrogen bondswith the water molecules, and therefore reduces an interaction betweenthe linker and the protein moieties. Rigid linkers generally maintainthe distance between the protein domains and they may be based onhelical structures and/or they have a sequence that is rich in praline.Cleavable linkers (also termed “cleavage linkers”) allow for in vivoseparation of the protein domains. The mechanism of cleavage may bebased e.g. on reduction of disulfide bonds within the linker sequence orproteolytic cleavage. The cleavage may be mediated by an enzyme(enzymatic cleavage), e.g. the cleavage linker may provide a proteasesensitive sequence (e.g., furin cleavage).

A typical sequence of a flexible linker is composed of repeats of theamino acids Glycine (G) and Serine (S). For instance, the linker mayhave the following sequence: GS, GSG, SGG, SG, GGS, SGS, GSS, SSG. Insome embodiments, the same sequence is repeated multiple times (e.g.two, three, four, five or six times) to create a longer linker. In otherembodiments, a single amino acid residue such as S or G can be used as alinker.

Linkers or spacers may be used as additional elements to promote orimprove the secretion of the target protein (Norovirus antigenicpeptides or proteins).

According to other embodiments, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one self-cleaving peptide.

Viral self-cleaving peptides (2A peptides) allow the expression ofmultiple proteins from a single open reading frame. The terms 2A peptideand 2A element are used interchangeably herein. The mechanism by the 2Asequence for generating two proteins from one transcript is by ribosomeskipping—a normal peptide bond is impaired at 2A, resulting in twodiscontinuous protein fragments from one translation event.

When used in the context of the present invention, such 2A peptides areparticularly useful when encoded by a nucleic acid encoding at least twofunctional protein elements (e.g. two Norovirus antigenic peptides orproteins). In general, a 2A element is useful when the nucleic acidmolecule encodes at least one polypeptide or protein of interest and atleast one further protein element. In a preferred embodiment, a 2Aelement is present when the polynucleotide of the invention encodes twoproteins or polypeptides of interest, e.g. two antigens.

The coding sequence for such 2A peptide is typically located in betweenthe coding sequence of the polypeptide of interest and the codingsequence of the least one further protein element (which may also be apolypeptide of interest), so that cleavage of the 2A peptide leads totwo separate polypeptide molecules, at least one of them being apolypeptide or protein of interest.

For example, for expressing target proteins (Norovirus antigenicpeptides or proteins) that are composed of several polypeptide chains itmay be beneficial to provide coding information for both polypeptidechains on a single nucleic acid molecule, separated by a nucleic acidsequence encoding a 2A peptide. 2A peptides may also be beneficial whencleavage of the protein of interest from another encoded polypeptideelement is desired.

2A peptides may be derived from foot-and-mouth diseases virus, fromequine rhinitis A virus, Thosea asigna virus, Porcine teschovirus-1.

According to other embodiments, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one helper peptide.

In essence, helper peptides binds to class II MHC molecules as anonspecific vaccine helper epitope (adjuvant) and induces an increased(and long term) immune response by increasing the helper T-cellresponse. In an embodiment, such a helper peptide may be N-terminallyand/or C-terminally fused to the antigenic peptide or protein derivedfrom Norovirus.

mRNA Structures:

Any of the above modifications may be applied to the artificial nucleicacid of the present invention, and further to any nucleic acid as usedin the context of the present invention and may be, if suitable ornecessary, be combined with each other in any combination, provided,these combinations of modifications do not interfere with each other inthe artificial nucleic acid. A person skilled in the art will be able totake his choice accordingly.

The artificial nucleic acid as defined herein, may preferably comprise a5′-UTR, a coding region encoding the at least one polypeptide comprisingat least one Norovirus protein as described herein, or a fragment,variant or derivative thereof; and/or a 3′-UTR preferably containing atleast one histone stem-loop. The 3′-UTR of the artificial nucleic acidpreferably comprises also a poly(A) and/or a poly(C) sequence as definedherewithin. The single elements of the 3′-UTR may occur therein in anyorder from 5′ to 3′ along the sequence of the artificial nucleic acid.In addition, further elements as described herein, may also becontained, such as a stabilizing sequence as defined herewithin (e.g.derived from the UTR of a globin gene), IRES sequences, etc. Each of theelements may also be repeated in the artificial nucleic acid accordingto the invention at least once (particularly in di- or multicistronicconstructs), preferably twice or more. As an example, the singleelements may be present in the artificial nucleic acid in the followingorder:

5′-coding region-histone stem-loop-poly(A)/(C) sequence-3′; or5′-coding region-poly(A)/(C) sequence-histone stem-loop-3′; or5′-coding region-histone stem-loop-polyadenylation signal-3′; or5′-coding region-polyadenylation signal-histone stem-loop-3′; or5′-coding region-histone stem-loop-histone stem-loop-poly(A)/(C)sequence-3′; or5′-coding region-histone stem-loop-histone stem-loop-polyadenylationsignal-3′; or5′-coding region-stabilizing sequence-poly(A)/(C) sequence-histonestem-loop-3′; or5′-coding region-stabilizing sequence-poly(A)/(C) sequence-poly(A)/(C)sequence-histone stem-loop-3′; etc.

In this context, it is particularly preferred that if, in addition tothe at least one encoded polypeptide defined herein, a further peptideor protein is encoded by the artificial nucleic acid the encoded peptideor protein is preferably no histone protein, no reporter protein (e.g.Luciferase, GFP, EGFP, β-Galactosidase, particularly EGFP) and/or nomarker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:Guanine phosphoribosyl transferase (GPT)). In a preferredembodiment, the artificial nucleic acid according to the invention doesnot comprise a reporter gene or a marker gene. Preferably, theartificial nucleic acid according to the invention does not encode, forinstance, luciferase; green fluorescent protein (GFP) and its variants(such as eGFP, RFP or BFP); α-globin; hypoxanthine-guaninephosphoribosyltransferase (HGPRT); β-galactosidase; galactokinase;alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP)) ora resistance gene (such as a resistance gene against neomycin,puromycin, hygromycin and zeocin). In a preferred embodiment, theartificial nucleic acid according to the invention does not encodeluciferase. In another embodiment, the artificial nucleic acid accordingto the invention does not encode GFP or a variant thereof.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of, preferably in 5′ to 3′ direction, thefollowing elements:

-   -   a) optionally, a 5′-cap structure (cap0, cap1, cap2), preferably        m7GpppN,    -   b) a coding region encoding at least one protein comprising at        least one Norovirus protein as described herein, or a fragment        or variant thereof,    -   c) optionally a poly(A) tail, preferably consisting of 10 to        200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides,    -   d) optionally a poly(C) tail, preferably consisting of 10 to        200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine        nucleotides, and    -   e) optionally a histone stem-loop, preferably comprising the RNA        sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.

More preferably, the artificial nucleic acid according to the inventioncomprises or consists of, preferably in 5′ to 3′ direction, thefollowing elements:

-   -   a) optionally, a 5′-cap structure (cap0, cap1, cap2), preferably        m7GpppN,    -   b) a coding region encoding at least one protein comprising at        least one Norovirus protein as described herein, or a fragment        or variant thereof,    -   c) a 3′-UTR element comprising a nucleic acid sequence, which is        derived from an α-globin gene, preferably comprising the        corresponding RNA sequence of the nucleic acid sequence        according to SEQ ID NO: 39701, or SEQ ID NO: 39702, or a        homolog, a fragment or a variant thereof,    -   d) optionally a poly(A) tail, preferably consisting of 10 to        200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides,    -   e) optionally a poly(C) tail, preferably consisting of 10 to        200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine        nucleotides, and    -   f) optionally a histone stem-loop, preferably comprising the RNA        sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.

According to a particularly preferred embodiment the RNA sequenceaccording to the invention comprises, preferably in 5′- to 3′-direction:

-   -   a) a 5′-cap structure (cap0, cap1, cap2), preferably m7GpppN;    -   b) a 5′-UTR element which comprises or consists of a nucleic        acid sequence corresponding to a nucleic acid sequence according        to SEQ ID NO: 39691, or SEQ ID NO: 39692, or SEQ ID NO: 39693,        or SEQ ID NO: 39694, a homolog, a fragment or a variant thereof;    -   c) at least one coding sequence according to any one of SEQ ID        NOs: 8821-39690, 39717, and/or 39730 or a variant or fragment        thereof encoding at least one antigenic peptide or protein        derived from a Norovirus protein or peptide or a fragment or        variant thereof according to any one of SEQ ID NOs: 1-4410,        preferably comprising or consisting of any one of the nucleic        acid sequences according to, or a fragment or variant thereof,    -   d) a 3′-UTR element comprising or consisting of a nucleic acid        sequence which is derived from a gene providing a stable RNA,        preferably comprising or consisting of the corresponding to a        nucleic acid sequence according to SEQ ID NO: 39707, or SEQ ID        NO: 39708, or SEQ ID NO: 39703, or SEQ ID NO: 39704, a homolog,        a fragment or a variant thereof;    -   e) optionally, a poly(A) sequence preferably comprising 64        adenosines; and    -   f) optionally, a poly(C) sequence, preferably comprising 3D        cytosines.

More preferably, the artificial nucleic acid according to the inventioncomprises or consists of, preferably in 5′ to 3′ direction, thefollowing elements:

-   -   a) optionally, a 5′-cap structure (cap0, cant cap2), preferably        m7GpppN,    -   b) a 5′-UTR element, which comprises or consists of a nucleic        acid sequence, which is derived from the 5′-UTR of a TOP gene,        preferably comprising a nucleic acid sequence according to SEQ        ID NO: 39691 to SEQ ID NO: 39694, or a homolog, a fragment or a        variant thereof,    -   c) a coding sequence encoding at least one protein comprising at        least one Norovirus protein as described herein, or a fragment        or variant thereof,    -   d) a 3′-UTR element comprising a nucleic acid sequence, which is        derived from an albumin gene, preferably comprising the        corresponding RNA sequence of the nucleic acid sequence        according to SEQ ID NO: 39705, or SEQ ID NO: 39706, or a        homolog, a fragment or a variant thereof,    -   e) optionally a poly(A) tail, preferably consisting of 10 to        200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides,    -   f) optionally a poly(S) tail, preferably consisting of 10 to        200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine        nucleotides, and    -   g) optionally a histone stem-loop, preferably comprising the RNA        sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.

In some embodiments, the at least one coding region of the artificialnucleic acid according to the present invention comprises a nucleic acidsequence encoding a molecular tag. More preferably, the molecular tag isselected from the group consisting of a FLAG tag, aglutathione-S-transferase (GST) tag, a His tag, a Myc tag, an E tag, aStrep tag, a green fluorescent protein (GFP) tag and an HA tag.

In particularly preferred embodiments the mRNA sequence according to theinvention comprises the following mRNA sequences (or RNA sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thefollowing RNA sequences):

-   -   mRNA encoding VP1 protein of Norovirus GII.4.1131693-USA-2003;        SEQ ID NO: 39713.    -   mRNA encoding VP1 protein of Norovirus GII.4-031693-USA-2003;        SEQ ID NO: 39714.    -   mRNA encoding VP1 protein of Norovirus GII.4-031693-USA-2003;        SEQ ID NO: 39715.    -   mRNA encoding VP1 protein of Norovirus GII.4-031693-USA-2003;        SEQ ID NO: 39716.    -   mRNA encoding VP1 protein of Norovirus GII.4-031693-USA-2003;        SEQ ID NO: 39717.    -   mRNA encoding VP1 protein of Norovirus GII.4 Farmington        Hills-2002-USA; SEQ ID NO: 39718.    -   mRNA encoding VP1 protein of Norovirus GII.4 Farmington        Hills-2002-USA; SEQ ID NO: 39719.    -   mRNA encoding VP1 protein of Norovirus GII.4 Farmington        Hills-2002-USA; SEQ ID NO: 39720.    -   mRNA encoding VP1 protein of Norovirus GII.4 Farmington        Hills-2002-USA; SEQ ID NO: 39721.    -   mRNA encoding VP1 protein of Norovirus GI.1-USA-1968; SEQ ID NO:        39722.    -   mRNA encoding VP1 protein of Norovirus GI.1-USA-1968; SEQ ID NO:        39723.    -   mRNA encoding VP1 protein of Norovirus GI.1-USA-1968; SEQ ID NO:        39724.    -   mRNA encoding VP1 protein of Norovirus GI.1-USA-1968; SEQ ID NO:        39725.    -   mRNA encoding VP1 protein of Norovirus GII.4 2006b        092895-USA-2008; SEQ ID NO: 39726.    -   mRNA encoding VP1 protein of Norovirus GII.4 2006b        092895-USA-2008; SEQ ID NO: 39727.    -   mRNA encoding VP1 protein of Norovirus GII.4 2006b        092895-USA-2008; SEQ ID NO: 39728.    -   mRNA encoding VP1 protein of Norovirus GII.4 2006b        092895-USA-2008; SEQ ID NO: 39729.    -   mRNA encoding VP1 protein of Norovirus GII.4 2006b        092895-USA-2008; SEQ ID NO: 39730.    -   mRNA encoding VP1 protein of Norovirus GII.4        GZ2010-L87-Guangzhou-2011; SEQ ID NO: 39731.    -   mRNA encoding VP1 protein of Norovirus GII.4        GZ2010-L87-Guangzhou-2011; SEQ ID NO: 39732.    -   mRNA encoding VP1 protein of Norovirus GII.4        GZ2010-L87-Guangzhou-2011; SEQ ID NO: 39733.    -   mRNA encoding VP1 protein of Norovirus GII.4        G2010-L87-Guangzhou-2011; SEQ ID NO: 39734.    -   mRNA encoding VP1 protein of Norovirus GII.4 USA-1997; SEQ ID        NO: 39735.    -   mRNA encoding VP1 protein of Norovirus GII.4 USA-1997: SEQ ID        NO: 39736.    -   mRNA encoding VP1 protein of Norovirus GII.4 USA-1997; SEQ ID        NO: 39737.    -   mRNA encoding VP1 protein of Norovirus GII.4 USA-1997; SEQ ID        NO: 39738.    -   mRNA encoding VP1 protein of Norovirus Melksham; SEQ ID NO:        39739.    -   mRNA encoding VP1 protein of Norovirus Melksham; SEQ ID NO:        39740.    -   mRNA encoding VP1 protein of Norovirus Melksham; SEQ ID NO:        39741.    -   mRNA encoding VP1 protein of Norovirus Melksham; SEQ ID NO:        39742.    -   mRNA encoding VP1 protein of Norovirus GII.2-Vaals87-2005-NL;        SEQ ID NO: 39743.    -   mRNA encoding VP1 protein of Norovirus GII.2-Vaals87-2005-NL;        SEQ ID NO: 39744.    -   mRNA encoding VP1 protein of Norovirus GII.2-Vaals87-2005-NL;        SEQ ID NO: 39745.    -   mRNA encoding VP1 protein of Norovirus GII.2-Vaals87-2005-NL;        SEQ ID NO: 39746.

RNA Production:

The artificial nucleic acid according to the invention may be preparedby using any suitable method known in the art, including syntheticmethods such as e.g. solid phase synthesis, as well as recombinant andin vitro methods, such as in vitro transcription reactions.

In a preferred embodiment, a linear DNA template is transcribed in vitrousing DNA dependent T7 RNA polymerase in the presence of a nucleotidemixture and cap analog (m7GpppG) under suitable buffer conditions. In aparticularly preferred embodiment, RNA production is performed undercurrent good manufacturing practice, implementing various qualitycontrol steps, e.g. according to WO 2016/180430. The obtained RNAs areHPLC purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008/077592). In a preferred embodiment, purified RNA product islyophilized according to WO 2016/165831 to yield a temperature stableNorovirus artificial nucleic acid. For the production of polyvalentNorovirus compositions, methods as disclosed in the PCT applicationPCT/EP2016/082487 are preferably used and adapted accordingly.

Composition:

In a further aspect, the present invention provides a compositioncomprising at least one artificial nucleic acid as described herein anda suitable carrier, preferably a pharmaceutically acceptable carrier.The inventive composition comprising the artificial nucleic acid asdescribed herein is preferably a (pharmaceutical) composition or avaccine as described herein.

The inventive composition may comprise either only one type ofartificial nucleic acid or at least two different artificial nucleicacids. In particular, the inventive composition may comprise at leasttwo artificial nucleic acids as described herein, wherein each of the atleast two artificial nucleic acids comprises at least one coding regionencoding at least one polypeptide comprising a different one of theNorovirus proteins as described herein, or a fragment or a variant ofany one of these proteins. Alternatively, the composition may compriseat least two artificial nucleic acids as described herein, wherein eachof the at least two artificial nucleic acids comprises at least onecoding region encoding at least one polypeptide comprising at least twodifferent Norovirus proteins as described herein, or a fragment or avariant of any one of these proteins. In another embodiment, thecomposition may also comprise at least two different artificial nucleicacids, which are bi- or multicistronic nucleic acids as described hereinand wherein each of the artificial nucleic acids encodes at least twopolypeptides, each comprising at least one Norovirus protein, or afragment or variant thereof.

Preferably, the inventive composition comprises or consists of at leastone artificial nucleic acid as described herein and a pharmaceuticallyacceptable carrier. The expression “pharmaceutically acceptable carrier”as used herein preferably includes the liquid or non-liquid basis of theinventive composition, which is preferably a pharmaceutical compositionor a vaccine. If the inventive composition is provided in liquid form,the carrier will preferably be water, typically pyrogen-free water;isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrateetc. buffered solutions. Water or preferably a buffer, more preferablyan aqueous buffer, may be used, containing a sodium salt, preferably atleast 50 mM of a sodium salt, a calcium salt, preferably at least 0.01mM of a calcium salt, and optionally a potassium salt, preferably atleast 3 mM of a potassium salt. According to a preferred embodiment, thesodium, calcium and, optionally, potassium salts may occur in the formof their halogenides, e.g. chlorides, iodides, or bromides, in the formof their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.Without being limited thereto, examples of sodium salts include e.g.NaCL, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of the optionalpotassium salts include e.g. KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, andexamples of calcium salts include e.g. CaCl₂, CaIz, CaBr₂, CaCO₃, CaSO₄,Ca(OH)₂. Furthermore, organic anions of the aforementioned cations maybe contained in the buffer.

Furthermore, one or more compatible solid or liquid fillers or diluentsor encapsulating compounds may be used as well, which are suitable foradministration to a person. The term “compatible” as used herein meansthat the constituents of the inventive composition are capable of beingmixed with the at least one artificial nucleic acid of the composition,in such a manner that no interaction occurs, which would substantiallyreduce the biological activity or the pharmaceutical effectiveness ofthe inventive composition under typical use conditions. Pharmaceuticallyacceptable carriers, fillers and diluents must, of course, havesufficiently high purity and sufficiently low toxicity to make themsuitable for administration to a person to be treated. Some examples ofcompounds which can be used as pharmaceutically acceptable carriers,fillers or constituents thereof are sugars, such as, for example,lactose, glucose, trehalose and sucrose; starches, such as, for example,corn starch or potato starch; dextrose; cellulose and its derivatives,such as, for example, sodium carboxymethylcellulose, ethylcellulose,cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solidgliddants, such as, for example, stearic acid, magnesium stearate;calcium sulfate; vegetable oils, such as, for example, groundnut oil,cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma;polyols, such as, for example, polypropylene glycol, glycerol, sorbitol,mannitol and polyethylene glycol; alginic acid.

Further additives which may be included in the inventive composition areemulsifiers, such as, far example, Tween; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

In another embodiment, the composition of the invention comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100 or more artificial nucleic acids ofthe invention, wherein each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more artificial nucleic acids of the invention comprises at least onecoding region encoding at least one polypeptide comprising a Norovirusprotein, and/or a fragment or a variant of any one of these proteins,wherein each coding region preferably encodes a different Norovirusprotein, more preferably each coding region encodes a capsid protein,preferably VP1 of a different Norovirus.

In another embodiment, the composition of the invention comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5051, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100 or more artificial nucleic acids ofthe invention, wherein each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 5051, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more artificial nucleic acids of the invention comprises at least onecoding region encoding at least one polypeptide comprising at least twodifferent Norovirus proteins, preferably VP1 and VP2, and/or a fragmentor a variant of any one of these proteins.

In a preferred embodiment, the inventive composition, which ispreferably a pharmaceutical composition or a vaccine, comprises at leastone artificial nucleic acid as described herein, wherein the at leastone artificial nucleic acid is complexed at least partially with acationic or polycationic compound and/or a polymeric carrier, preferablya cationic protein or peptide. Accordingly, in a further embodiment ofthe invention it is preferred that the at least one artificial nucleicacid as defined herein or any other nucleic acid comprised in theinventive (pharmaceutical) composition or vaccine is associated with orcomplexed with a cationic or polycationic compound or a polymericcarrier, optionally in a weight ratio selected from a range of about 6:1(w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) toabout 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1(w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably aratio of about 3:1 (w/w) to about 2:1 (w/w) of the artificial nucleicacid or any other nucleic acid to cationic or polycationic compoundand/or with a polymeric carrier: or optionally in a nitrogen/phosphate(NIP) ratio of the artificial nucleic acid or any other nucleic acid tocationic or polycationic compound and/or polymeric carrier in the rangeof about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and mostpreferably in a range of about 0.5-1 or 0.7-1, and even most preferablyin a range of about 0.3-0.9 or 0.5-0.9. More preferably, the N/P ratioof the at least one artificial nucleic acid to the one or morepolycations is in the range of about 0.1 to 10, including a range ofabout 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and of about 0.7 to1.5.

In another embodiment, the composition comprising at least oneartificial nucleic acid of the invention is defined as follows:

-   -   (i) the ratio of complexed nucleic acid to free nucleic acid is        selected from a range of about 5:1 (w/w) to about 1:10 (w/w),        more preferably from a range of about 4:1 (w/w) to about 1:3        (w/w), even more preferably from a range of about 3:1 (w/w) to        about 1:5 (w/w) or 1:3 (w/w), wherein the ratio is most        preferably about 1:1 (w/w): or    -   (ii) the mRNA is complexed with one or more cationic or        polycationic compounds in a weight ratio selected from a range        of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from        about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of        about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w) to        about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)        to about 2:1 (w/w) of mRNA to cationic or polycationic compound        and/or with a polymeric carrier: or optionally in a        nitrogen/phosphate ratio of mRNA to cationic or polycationic        compound and/or polymeric carrier in the range of about 0.1-10,        preferably in a range of about 0.3-4 or 0.3-1, and most        preferably in a range of about 0.5-1 or 0.7-1, and even most        preferably in a range of about 0.3-0.9 or 0.5-0.9;    -   and/or wherein the at least one artificial nucleic acid or mRNA        is complexed with one or more cationic or polycationic        compounds, preferably with cationic or polycationic polymers,        cationic or polycationic peptides or proteins, e.g. protamine,        cationic or polycationic polysaccharides and/or cationic or        polycationic lipids and/or wherein the at least one artificial        nucleic acid or mRNA is complexed with one or more lipids and        thereby forming liposomes, lipid nanoparticles and/or        lipoplexes.

Preferably, the inventive composition comprises at least one artificialnucleic acid as described herein, which is complexed with one or morepolycations and/or a polymeric carrier, and at least one free nucleicacid, wherein the at least one complexed nucleic acid is preferablyidentical to the at least one artificial nucleic acid according to thepresent invention. In this context it is particularly preferred that theat least one artificial nucleic acid of the inventive composition iscomplexed at least partially with a cationic or polycationic compoundand/or a polymeric carrier, preferably cationic proteins or peptides. Inthis context, the disclosure of WO 2010/037539 and WO 2012/113513 isincorporated herewith by reference. Partially means that only a part ofthe inventive artificial nucleic acid is complexed with a cationiccompound and that the rest of the inventive artificial nucleic acid is(comprised in the inventive pharmaceutical composition or vaccine) inuncomplexed form (“free”).

In a further embodiment, the composition of the invention comprises

-   -   (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more artificial        nucleic acids as defined in the invention; or    -   (ii) at least 10, 15, 20 or 50 artificial nucleic acids as        defined in the invention; or    -   (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificial        nucleic acids as defined in the invention; and a        pharmaceutically acceptable carrier.

In one embodiment, the compositions and/or vaccines of the inventioncomprise artificial nucleic acids encoding one or more capsid proteinsVP1 derived from one or more Noroviruses. In another embodiment, thecompositions and/or vaccines of the invention comprise artificialnucleic acids encoding one or more capsid proteins VP2 derived from oneor more Noroviruses.

In another embodiment, the composition of the invention is furtherdefined as composition, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 50, 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,        78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,        94, 95, 96, 97, 98, 99, 100 or more different GI Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single GII        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 56, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or more different GIII Noroviruses; or    -   (iii) the artificial nucleic acids are derived from a single        GIII Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,        78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,        94, 95, 96, 97, 98, 99, 100 or mare different GIII Noroviruses;        or    -   (iv) the artificial nucleic acids are derived from a single GIV        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or more different GIV Noroviruses; or    -   (v) the artificial nucleic acids are derived from a single GV        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 59, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or more different GV Noroviruses; or    -   (vi) the artificial nucleic acids are derived from a single GI        Norovirus and additionally from a single GII Norovirus, GIII        Norovirus, GIV Norovirus and/or GU Norovirus; or    -   (vii) the artificial nucleic acids are derived from a single GI        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or mare different GI Noroviruses and        additionally from a single 911, GIII, GIV or GV Norovirus and/or        from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,        35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,        51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,        67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,        83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,        99, 100 and/or mare GII, GIII, GIV or GV Noroviruses.

In a further embodiment, the composition of the invention further isdefined as composition, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or more different GI.1 Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single        GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,        78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,        94, 95, 96, 97, 98, 99, 100 or more different GII.4 Noroviruses;        or    -   (iii) the artificial nucleic acids are derived from a single        GI.1 Norovirus and additionally from a single GII.4 Norovirus;        or    -   (iv) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or more different GI.1 Noroviruses and        additionally from a single GII.4 Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        GII.4 Noroviruses.

In a further embodiment, the composition of the invention further isdefined as composition, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or mare different GII.4 Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single        GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,        78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,        94, 95, 96, 97, 98, 99, 100 or more different GII.4 Noroviruses;        or    -   (iii) the artificial nucleic acids are derived from a single        GI.1 Norovirus and additionally from a single GII.4 Norovirus;        or    -   (iv) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,        63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,        79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,        95, 96, 97, 98, 99, 100 or more different GI.1 Noroviruses and        additionally from a single GII.4 Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        GII.4 Noroviruses; and/or    -   wherein    -   (i) at least one of the nucleic acid sequences according to any        one of SEQ ID NOs: 4411-39690, 39713-39746; and/or (ii) at least        one of the nucleic acid sequences having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the nucleic acid sequence        represented by any one of SEQ ID NOs: 4411-39690, 39713-39746;        and/or    -   (iii) at least one complement of the nucleic acid sequences        which are capable of hybridizing with a nucleic acid sequence        comprising a sequence as shown in SEQ ID NDs: 4411-39690,        39713-39746, and/or    -   (iv) an orthologue or a paralogue of any one of SEQ ID NOs:        4411-39690, 39713-39746; and/or a fragment or variant of any of        these sequences.    -   and/or    -   wherein    -   (i) at least one of the nucleic acid sequences according to any        one of SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or    -   (ii) at least one of the nucleic acid sequences having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 68%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        nucleic acid sequence represented by any one of SEQ ID NO: 39713        to SEQ ID NO: 39746; and/or    -   (iii) at least one complement of the nucleic acid sequences        which are capable of hybridizing with a nucleic acid sequence        comprising a sequence as shown in SEQ ID NO: 39713 to SEQ ID NO:        39746; and/or    -   (iv) an orthologue or a paralogue of any one of SEQ ID NO: 39713        to SEQ ID NO: 39746; and/or a fragment or variant of any of        these sequences.

In a preferred embodiment, the composition of the invention further isdefined as a composition comprising two different nucleic acid sequences(bivalent, divalent composition), wherein one of the two differentnucleic acid sequences is derived from Norovirus GII.4 and one of thetwo different nucleic acid sequence is derived from Norovirus GI.1,wherein the one nucleic acid sequence of Norovirus 811.4 may be any oneof the nucleic acid sequences as defined herein and in Table 1, orfragment or variants of these sequences, and wherein the one nucleicacid sequence of Norovirus GI.1 may be any one of the nucleic acidsequences as defined herein and in Table 1, or fragment or variants ofthese sequences. Preferably, the composition comprising two differentnucleic acid sequences (bivalent, divalent composition) comprises orconsists of one nucleic acid sequence derived from Norovirus GII.4selected from SEQ ID NOs: 39713-39721, 39726-39742, and one nucleic acidsequence derived from Norovirus GI.1 selected from SEQ ID NDs:39722-39725.

In a specific preferred embodiment, the composition comprising twodifferent nucleic acid sequences (bivalent, divalent composition)comprises or consists of the nucleic acid sequences SEQ ID NO: 39716(Norovirus GII.4) and SEQ ID NO: 39725 (Norovirus GI.1). In a furtherspecific preferred embodiment, the composition comprising two differentnucleic acid sequences (bivalent, divalent composition) comprises orconsists of the nucleic acid sequences SEQ ID NO: 39721 (NorovirusGII.4) and SEQ ID NO: 39725 (Norovirus GI.1). In a further specificpreferred embodiment, the composition comprising two different nucleicacid sequences (bivalent, divalent composition) comprises or consists ofthe nucleic acid sequences SEQ ID NO: 39729 (Norovirus GII.4) and SEQ IDNO: 39725 (Norovirus GI.1). In a further specific preferred embodiment,the composition comprising two different nucleic acid sequences(bivalent, divalent composition) comprises or consists of the nucleicacid sequences SEQ ID NO: 39734 (Norovirus GII.4) and SEQ ID NO: 39725(Norovirus GI.1). In a further specific preferred embodiment, thecomposition comprising two different nucleic acid sequences (bivalent,divalent composition) comprises or consists of the nucleic acidsequences SEQ ID NO: 39738 (Norovirus 811.4) and SEQ ID NO: 39725(Norovirus GI.1).

In a preferred embodiment, the composition of the invention further isdefined as a composition comprising two different nucleic acid sequences(bivalent, divalent composition) derived from Norovirus GII.4, whereinthe two different nucleic acid sequences of Norovirus GII.4 may be anyone of the nucleic acid sequences as defined herein and in Table 1, orfragment or variants of these sequences. Preferably, the compositioncomprising two different nucleic acid sequences derived from NorovirusGII.4 (bivalent, divalent composition) comprises or consists of onenucleic acid sequence selected from SEQ ID NOs: 39713-39721,39726-39742.

In a specific preferred embodiment, the composition comprising twodifferent nucleic acid sequences (bivalent, divalent composition)comprises or consists of the nucleic acid sequences SEQ ID NO: 39716(Norovirus GII.4) and SEQ ID NO: 39721 (Norovirus GII.4). In a specificpreferred embodiment, the composition comprising two different nucleicacid sequences (bivalent, divalent composition) comprises or consists ofthe nucleic acid sequences SEQ ID NO: 39729 (Norovirus GII.4) and SEQ IDNO: 39721 (Norovirus GII.4). In a specific preferred embodiment, thecomposition comprising two different nucleic acid sequences (bivalent,divalent composition) comprises or consists of the nucleic acidsequences SEQ ID NO: 39734 (Norovirus GII.4) and SEQ ID NO: 39721(Norovirus GII.4). In a specific preferred embodiment, the compositioncomprising two different nucleic acid sequences (bivalent, divalentcomposition) comprises or consists of the nucleic acid sequences SEQ IDNO: 39738 (Norovirus GII.4) and SEQ ID NO: 39721 (Norovirus GII.4). In aspecific preferred embodiment, the composition comprising two differentnucleic acid sequences (bivalent, divalent composition) comprises orconsists of the nucleic acid sequences SEQ ID NO: 39716 (NorovirusGII.4) and SEQ ID NO: 39729 (Norovirus GII.4). In a specific preferredembodiment, the composition comprising two different nucleic acidsequences (bivalent, divalent composition) comprises or consists of thenucleic acid sequences SEQ ID NO: 39734 (Norovirus GII.4) and SEQ ID NO:39729 (Norovirus GII.4). In a specific preferred embodiment, thecomposition comprising two different nucleic acid sequences (bivalent,divalent composition) comprises or consists of the nucleic acidsequences SEQ ID NO: 39738 (Norovirus GII.4) and SEQ ID NO: 39729(Norovirus GII.4). In a specific preferred embodiment, the compositioncomprising two different nucleic acid sequences (bivalent, divalentcomposition) comprises or consists of the nucleic acid sequences SEQ IDNO: 39719 (Norovirus GII.4) and SEQ ID NO: 39734 (Norovirus GII.4). In aspecific preferred embodiment, the composition comprising two differentnucleic acid sequences (bivalent, divalent composition) comprises orconsists of the nucleic acid sequences SEQ ID NO: 39738 (NorovirusGII.4) and SEQ ID NO: 39734 (Norovirus GII.4). In a specific preferredembodiment, the composition comprising two different nucleic acidsequences (bivalent, divalent composition) comprises or consists of thenucleic acid sequences SEQ ID NO: 39719 (Norovirus GII.4) and SEQ ID NO:39738 (Norovirus GII.4).

In further a preferred embodiment, the composition of the inventionfurther is defined as a composition comprising four different nucleicacid sequences (tetravalent composition), wherein each of the fourdifferent nucleic acid sequences is derived from Norovirus GII.4,wherein each of the four different nucleic of Norovirus GII.4 may be anyone of the nucleic acid sequences as defined herein and in Table 1, orfragment or variants of these sequences. Preferably, the compositioncomprising four different nucleic acid sequences (tetravalentcomposition) comprises or consists of four nucleic acid sequence derivedfrom Norovirus GII.4 selected from SEQ ID NOs: 39713-39721, 39726-39742.

In a specific preferred embodiment, the composition comprising fourdifferent nucleic acid sequences (tetravalent composition) comprisesfour of the nucleic acid sequences selected from SEQ ID NOs: 39716,39721, 39729, 39734 or 39738.

In a further preferred embodiment, the composition of the invention isdefined as a composition comprising four different nucleic acidsequences (tetravalent composition), wherein at least one of the fourdifferent nucleic acid sequences is derived from Norovirus GII.4 and atleast one of the four different nucleic acid sequence is derived fromNorovirus GI.1, wherein the at least one of the four different nucleicof Norovirus GII.4 may be any one of the nucleic acid sequences asdefined herein and in Table 1, or fragment or variants of thesesequences, and wherein the at least one of the four of nucleic acidsequence of Norovirus GI.1 may be any one of the nucleic acid sequencesas defined herein and in Table 1, or fragment or variants of thesesequences.

In a further preferred embodiment, the composition of the invention isdefined as a composition comprising four different nucleic acidsequences (tetravalent composition), wherein three of the four differentnucleic acid sequences are derived from Norovirus GII.4 and one of thefour different nucleic acid sequence is derived from Norovirus GI.1,wherein the three of the four different nucleic of Norovirus GII.4 maybe any one of the nucleic acid sequences as defined in Table 1, orfragment or variants of these sequences, and wherein the one of the fourof nucleic acid sequence of Norovirus GI.1 may be any one of the nucleicacid sequences as defined in Table 1, or fragment or variants of thesesequences. Preferably, the composition comprising four different nucleicacid sequences (tetravalent composition) comprises or consists of threenucleic acid sequence derived from Norovirus GII.4 selected from SEQ IDNOs: 39713-39721, 39726-39742 and one nucleic acid sequence derived fromNorovirus GI.1 SEQ ID NOs: 39722-39725.

In a specific preferred embodiment, the composition comprising fourdifferent nucleic acid sequences (tetravalent composition) comprisesthree of the nucleic acid sequences derived from Norovirus GII.4selected from SEQ ID NOs: 39716, 39721, 39729, 39734 or 39738 and onenucleic acid sequence derived from Norovirus GI.1 SEQ ID NO: 39725.

In further a preferred embodiments, the composition of the invention isdefined as a composition comprising multiple different nucleic acidsequences (multivalent composition) defined as a composition comprising5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100 different nucleic acid sequences derived fromNorovirus GI.1, Norovirus GI.2, Norovirus GI.3, Norovirus GI.4,Norovirus GI.5, Norovirus GI.6, Norovirus GI.7, Norovirus GI.8,Norovirus GI.9, Norovirus GII.1, Norovirus GII.2, Norovirus GII.3,Norovirus GII.4, Norovirus GII.5, Norovirus GII.6, Norovirus GII.7,Norovirus GII.11, Norovirus GII.12, Norovirus GII.13, Norovirus GII.14,Norovirus GII.15, Norovirus GII.16, Norovirus GII.17, Norovirus GII.21,Norovirus GIII.1, Norovirus GV.1 or Norovirus GIV.1.

In further a preferred embodiments, the composition of the invention isdefined as a composition comprising multiple different nucleic acidsequences (multivalent composition) defined as a composition comprising5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100 different nucleic acid sequences selected from Table3 (column 4 and column 5). In Table 3, each row (row 1-row 138)corresponds to selected Norovirus protein or antigen as identified bythe respective name (first column, column 1 “Strain/Isolate”) and thedatabase accession number of the corresponding protein (second column,column 2 “NCBI or Genbank Accession No.”). The third column, column 3(“A”) in Table 3 indicates the SEQ ID NOs corresponding to therespective amino acid sequence as provided herein. The SEQ ID NOscorresponding to the nucleic acid sequence of the wild type nucleic acidsequence encoding the Norovirus protein or peptide is indicated in thefourth column, column 4 (“B”). The fifth column, column 5 (“C”) providesthe SEQ ID NOs corresponding to modified nucleic acid sequences of thenucleic acid sequences as described herein that encode the Norovirusprotein or peptide preferably having the amino acid sequence as definedby the SEQ ID NOs indicated in the third column (“A”) or by the databaseentry indicated in the second column (“14981 or Genbank Accession No.”).

TABLE 3 Selected VP1 Norovirus sequences (row 1-row 138) column 2 NCBIor Genbank column 1 Accession column 3 column 4 column 5 RowStrain/Isolate No. A B C 1 Norovirus Hu/GII.4/Dijon/E872/2002/FRAAC055068 2 4412 8822, 13232, 17642, 22052, 26462, 30872, 35282 2Norovirus Hu/GII.4/MD120-12/1987/USA AFS33552 16 4426 8836, 13246,17656, 22066, 26476, 30886, 35296 3 NorovirusHu/GII.1/7EK/Hawaii/1971/USA AFS33555 17 4427 8837, 13247, 17657, 22067,26477, 30887, 35297 4 Norovirus Hu/GII.6/CHDC4073/1984/USA AFX71665 274437 8847, 13257, 17667, 22077, 26487, 30897, 35307 5 NorovirusHu/GII.4/Hiroshima/19/2001/JPN BAI49904 327 4737 9147, 13557, 17967,22377, 26787, 31197, 35607 6 Norovirus Hu/GII.4/Hiroshima/67/2006/JPNBAI49914 335 4745 9155, 13565, 17975, 22385, 26795, 31205, 35615 7Norovirus Hu/GII/JP/2015/GII.Pe_GII.4/Osaka/OSF78 BAS02083 364 47749184, 13594, 18004, 22414, 26824, 31234, 35644 8 NorovirusGII/Hu/NL/2012/GII.4/Groningen CRL46958 367 4777 9187, 13597, 18007,22417, 26827, 31237, 35647 9 Norovirus GII/Hu/NL/2014/GII.2/GroningenCRL46973 372 4782 9192, 13602, 18012, 22422, 26832, 31242, 35652 10Norovirus Hu/GII.4/New Orleans1500/2008/USA ADB27027 376 4786 9196,13606, 18016, 22426, 26836, 31246, 35656 11 NorovirusHu/GII.6/Ohio/490/2012/USA AGI96397 386 4796 9206, 13616, 18026, 22436,26846, 31256, 35666 12 Norovirus Hu/GII.3/Jingzhou/2013402/CHN AGX01095391 4801 9211, 13621, 18031, 22441, 26851, 31261, 35671 13 NorovirusHu/GII.4/Jingzhou/2013403/CHN AGX01098 392 4802 9212, 13622, 18032,22442, 26852, 31262, 35672 14 Norovirus Hu/GII.17/Gaithersburg/2014/USAKI30060 393 4803 9213, 13623, 18033, 22443, 26853, 31263, 35673 15Norovirus Hu/GII.4/C127/GF/1978 AGL98413 404 4814 9224, 13634, 18044,22454, 26864, 31274, 35684 16 Norovirus Hu/GII.4/CHDC3967/1988/USACT76145 458 4868 9278, 13688, 18098, 22508, 26918, 31328, 35738 17Norovirus Hu/GII.4/CHDC4108/1987/US ACT76148 459 4869 9279, 13689,18099, 22509, 26919, 31329, 35739 18 Norovirus Hu/GII.4/CHDC4871/1977/USACT76151 460 4870 9280, 13690, 18100, 22510, 26920, 31330, 35740 19Norovirus Hu/GII.3/CHDC5261/1990/US AED02034 501 4911 9321, 13731,18141, 22551, 26961, 31371, 35781 20 NorovirusHu/GII.3/Milwaukee009/2010/USA AEX10549 534 4944 9354, 13764, 18174,22584, 26994, 31404, 35814 21 Norovirus Hu/GII.4/Miranda/NSW817L/2010/AUAFJ2I448 597 5007 9417, 13827, 18237, 22647, 27057, 31467, 35877 22Norovirus Hu/GII.2/KL109/MY/1978 AFN06726 603 5013 9423, 13833, 18243,22653, 27063, 31473, 35883 23 Norovirus Hu/GII.14/HK74/CN/1978 AFN06727604 5014 9424, 13834, 18244, 22654, 27064, 31474, 35884 24 NorovirusHu/GII.7/HK4/CN/1976 AFN06731 608 5018 9428, 13838, 18248, 22658, 27068,31478, 35888 25 Norovirus Hu/GII.17/C142/GF/1978 AFN06732 609 5019 9429,13839, 18249, 22659, 27069, 31479, 35889 26 NorovirusHu/GII.5/C15/GF/1978 AFN06733 610 5020 9430, 13840, 18250, 22660, 27070,31480, 35890 27 Norovirus Hu/GI.5/E57/UG/1975 AFN06735 612 5022 9432,13842, 18252, 22662, 27072, 31482, 35892 28 NorovirusHu/GII.4/Randwick/NSW882J/2011/AU AFV08771 616 5026 9436, 13846, 18256,22666, 27076, 31486, 35896 29 Norovirus Hu/GII.4/Berowra/NSW767L/2012/AUAFV08777 618 5028 9438, 13848, 18258, 22668, 27078, 31488, 35898 30Norovirus Hu/GII.4/Sydney/NSW0514/2012/AU AFV08795 624 5034 9444, 13854,18264, 22674, 27084, 31494, 35904 31 Norovirus Hu/GII.4/HongKong/CUHK3630/2012/CHN AFX95940 625 5035 9445, 13855, 18265, 22675,27085, 31495, 35905 32 Norovirus Hu/GII.4/VP1172/Shanghai/2012/CHNAGI99552 629 5039 9449, 13859, 18269, 22679, 27089, 31499, 35909 33Norovirus Hu/GII-4/New Taipei/CGMH61/2012/TW AGK25912 644 5054 9464,13874, 18284, 22694, 27104, 31514, 35924 34 NorovirusGII/Hu/HKG/2013/GII.4/CUHK-NS-141 AID68581 859 5269 9679, 14089, 18499,22909, 27319, 31729, 36139 35 NorovirusGII/Hu/JP/2002/GII.P12_GII.13/Saitama/T80 AII73717 877 5287 9697, 14107,18517, 22927, 27337, 31747, 36157 36 NorovirusGII/Hu/JP/2001/GII.P12_GII.12/Saitama/T15 AII73735 883 5293 9703, 14113,18523, 22933, 27343, 31753, 36163 37 NorovirusGII/Hu/JP/2007/GII.P21_GII.21/Kawasaki/Y0284 AII73741 885 5295 9705,14115, 18525, 22935, 27345, 31755, 36165 38 NorovirusGII/Hu/JP/2007/GII.P15_GII.15/Sapporo/HK299 AII73759 891 5301 9711,14121, 18531, 22941, 27351, 31761, 36171 39 NorovirusGI/Hu/JP/2007/GI.P3_GI.3/Shimizu/KK2866 AII73765 893 5303 9713, 14123,18533, 22943, 27353, 31763, 36173 40 NorovirusGII/Hu/JP/2007/GII.P7_GII.14/Fukuoka/KK282 AII73780 898 5308 9718,14128, 18538, 22948, 27358, 31768, 36178 41 NorovirusGI/Hu/JP/2007/GI.P8_GI.8/Nagoya/KY531 AII73783 899 5309 9719, 14129,18539, 22949, 27359, 31769, 36179 42 Norovirus Hu/GII.4/SJTUH1/CHN/2014AIS40019 901 5311 9721, 14131, 18541, 22951, 27361, 31771, 36181 43Norovirus Hu/GII.4/variant Sydney 2012/FRA AIY27747 930 5340 9750,14160, 18570, 22980, 27390, 31800, 36210 44 NorovirusHu/GII-4/Hokkaido4/2006/JP BAG70437 1033 5443 9853, 14263, 18673, 23083,27493, 31903, 36313 45 Norovirus GIV/Hu/Jp/2001/GIV.1/OCO1017023BAU16306 1345 5755 10165, 14575, 18985, 23395, 27805, 32215, 36625 46Norovirus Hu/GII.4/Beijing/53671/2007/CHN ACY00615 1454 5864 10274,14684, 19094, 23504, 27914, 32324, 36734 47 NorovirusHu/II.4/2200661/HK/2010 ADK47170 1467 5877 10287, 14697, 19107, 23517,27927, 32337, 36747 48 Norovirus Hu/GII.4/Aichi368-14/2014 BAQ20801 14775887 10297, 14707, 19117, 23527, 27937, 32347, 36757 49 NorovirusHu/GII.4/Hunter 284E/040/AU AAZ31376 1552 5962 10372, 14782, 19192,23602, 28012, 32422, 36832 50 Norovirus Hu/GII-4/Osaka/1998/JPN ABI979811569 5979 10389, 14799, 19209, 23619, 28029, 32439, 36849 51 NorovirusHu/GI.1/P774.Delsjo2004/Gothenburg/Sweden ABW74128 1610 6020 10430,14840, 19250, 23660, 28070, 32480, 36890 52 Noroviruspig/GII.11/F18-10/2005/CAN ACC69023 1629 6039 10449, 14859, 19269,23679, 28089, 32499, 36909 53 Norovirus Hu/GII.4/Wellington/1995/USAACL27297 1670 6080 10490, 14900, 19310, 23720, 28130, 32540, 36950 54Norovirus Hu/GII.4/Henry/2000/USA ACL27298 1671 6081 10491, 14901,19311, 23721, 28131, 32541, 36951 55 Norovirus Hu/GII.4/SSCS/2005/USAACL27299 1672 6082 10492, 14902, 19312, 23722, 28132, 32542, 36952 56Norovirus GII/Hu/IN/2006/GII.P4_GII.4_Yerseke2006a/Pune-PC21 ACL313221680 6090 10500, 14910, 19320, 23730, 28140, 32550, 36960 57 NorovirusHu/GI.1/P7-587/2007/Stromstad/Sweden ACN32270 1692 6102 10512, 14922,19332, 23742, 28152, 32562, 36972 58 Norovirus Hu/GI.2/Leuven/2003/BELACU56258 1698 6108 10518, 14928, 19338, 23748, 28158, 32568, 36978 59Norovirus Hu/GII.7/NSW743L/2008/AUS ACX85810 1712 6122 10532.14942,19352, 23762, 28172, 32582, 36992 60 Norovirus Hu/GII.2/NF2002/USA/2002AFB18010 1731 6141 10551, 14961, 19371, 23781, 28191, 32601, 37011 61Norovirus Hu/GII.4/NF2003/USA/2003 AFB18013 1732 6142 10552, 14962,19372, 23782, 28192, 32602, 37012 62 Norovirus Hu/GII.3/1999 AFK758541739 6149 10559, 14969, 19379, 23789, 28199, 32609, 37019 63 NorovirusHu/GIV.1/Ahrenshoop246/DEU/2012 AFN61315 1740 6150 10560, 14970, 19380,23790, 28200, 32610, 37020 64 Norovirus Hu/GII.4/Xi'an/P19/2010/CHNAFQ00511 1741 6151 10561, 14971, 19381, 23791, 28201, 32611, 37021 65Norovirus Hu/GII.4/PA363/2011/ITA AHC12655 1770 6180 10590, 15000,19410, 23820, 28230, 32640, 37050 66 NorovirusHu/GII.4/P3/2012/Gothenburg/Sweden AHZ12912 1778 6188 10598, 15008,19418, 23828, 28238, 32648, 37058 67 NorovirusHu/GII.4/Tanger/TM687/2011/MAR AIC32559 1787 6197 10607, 15017, 19427,23837, 28247, 32657, 37067 68 Norovirus 12-X-2/2012/GII.P22/GII.5AID51489 1793 6203 10613, 15023, 19433, 23843, 28253, 32663, 37073 69Norovirus Hu/GII.4/Kobe034/2006/JP BAF45861 1826 6236 10646, 15056,19466, 23876, 28286, 32696, 37106 70 Norovirus Hu/GGII.4/Tiel001/1995/NLBAF74508 1827 6237 10647, 15057, 19467, 23877, 28287, 32697, 37107 71Norovirus Hu/GGII.4/DenHaag015/2000/NL BAF74509 1828 6238 10648, 15058,19468, 23878, 28288, 32698, 37108 72 NorovirusHu/GGII.4/Schiedam018/2001/NL BAF74512 1831 6241 10651, 15061, 19471,23881, 28291, 32701, 37111 73 Norovirus Hu/GGII.4/Apeldoorn023/2003/NLBAF74517 1836 6246 10656, 15066, 19476, 23886, 28296, 32706, 37116 74Norovirus Hu/GGII.4/Middelburg007/2004/NL BAF74521 1840 6250 10660,15070, 19480, 23890, 28300, 32710, 37120 75 NorovirusHu/GII-4/Matsudo/021071/2002/JP BAF95499 1847 6257 10667, 15077, 19487,23897, 28307, 32717, 37127 76 Norovirus Hu/GII-4/Kaiso/030556/2003/JPBAF95501 1848 6258 10668, 15078, 19488, 23898, 28308, 32718, 37128 77Norovirus Hu/GII-4/Awa/040354/2004/JP BAF95505 1850 6260 10670, 15080,19490, 23900, 28310, 32720, 37130 78 NorovirusHu/GII.4/Apeldoorn3l7/2007/NL BAG55289 1885 6295 10705, 15115, 19525,23935, 28345, 32755, 37165 79 Norovirus Hu/GII.2/Rotterdam39E/2002/NLBAG68713 1891 6301 10711, 15121, 19531, 23941, 28351, 32761, 37171 80Norovirus Hu/GII.4/RotterdamP200/2005/NL BAG68801 1897 6307 10717,15127, 19537, 23947, 28357, 32767, 37177 81 NorovirusHu/GII.4/Stockholm/19865/2008/SE BAH30707 1916 6326 10736, 15146, 19556,23966, 28376, 32786, 37196 82 Norovirus Hu/GII.6/OCO4062VLP/2004/JPBAL40873 1921 6331 10741, 15151, 19561, 23971, 28381, 32791, 37201 83Norovirus Hu/GII.4/HS194/2009/US ADB27914 1961 6371 10781, 15191, 19601,24011, 28421, 32831, 37241 84 Norovirus Hu/GII.12/HS210/2010/USAADT70684 1965 6375 10785, 15195, 19605, 24015, 28425, 32835, 37245 85Norovirus Hu/GI.1/8FIIa/1968/USA AFJ38516 1970 6380 10790, 15200, 19610,24020, 28430, 32840, 37250 86 Norovirus Hu/GII.4/CHDC5191/1974/USAAFJ38519 1971 6381 10791, 15201, 19611, 24021, 28431, 32841, 37251 87Norovirus Hu/GII.4/N76/2010/HuZhou AFW15943 1975 6385 10795, 15205,19615, 24025, 28435, 32845, 37255 88 Norovirus Hu/GII.6/S9c/1976/SENAGE99599 2000 6410 10820, 15230, 19640, 24050, 28460, 32870, 37280 89Norovirus Hu/GII.4/KL45/1978/MYS AGE99612 2001 6411 10821, 15231, 19641,24051, 28461, 32871, 37281 90 Norovirus Hu/GII.4/NIHIC17.5/2012/USAAGT17839 2005 6415 10825, 15235, 19645, 24055, 28465, 32875, 37285 91Norovirus Hu/GII.4/NIHIC9/2011/USA AFX71669 4198 8608 13018, 17428,21838, 26248, 30658, 35068, 39478 92 Norovirus Hu/GII.4/C110/1978/GUFAGE99607 4336 8746 13156, 17566, 21976, 26386, 30796, 35206, 39616 93Norovirus Hu/GII.4/HS66/2001/USA AHI59166 4379 8789 13199, 17609, 22019,26429, 30839, 35249, 39659 94 NorovirusHu/GII/JP/2015/GII.P17_GII.17/Kawasaki308 BAR42290 4395 8805 13215,17625, 22035, 26445, 30855, 35265, 39675 95 NorovirusHu/GII/JP/2014/GII.P17_GII.17/Nagano8-1 BAR63722 4399 8809 13219, 17629,22039, 26449, 30859, 35269, 39679 96 NorovirusHu/GII/JP/2015/GII.Pe_GII.4/Osaka/OSF78 BAS02084 4400 8810 13220, 17630,22040, 26450, 30860, 35270, 39680 97 NorovirusGI/Hu/NL/2011/GI.4/Groningan CRL46953 4401 8811 13221, 17631, 22041,26451, 30861, 35271, 39681 98 Norovirus GII/Hu/NL/20l4/GII.4/Groningen01CRL46962 4404 8814 13224, 17634, 22044, 26454, 30864, 35274, 39684 99Norovirus Hu/GII.4/Kenepuru/NZ327/2006/NZL ABQ63283 2018 6428 10838,15248, 19658, 24068, 28478, 32888, 37298 100 NorovirusHu/GII.4/Rathmines/NSW287R/2007/AUS ACW19927 2023 6433 10843, 15253,19663, 24073, 28483, 32893, 37303 101 NorovirusHu/GII.4/Turramurra/NSW892U/2009/AUS ADQ43783 2032 6442 10852, 15262,19672, 24082, 28492, 32902, 37312 102 NorovirusHu/GII.4/Seoul/0389/2009/KOR ADV37805 2033 6443 10853, 15263, 19673,24083, 28493, 32903, 37313 103 Norovirus Hu/GII.4/Seoul/0945/2009/KORADV37919 2038 6448 10858, 15268, 19678, 24088, 28498, 32908, 37318 104Norovirus Hu/GII.12/Shelby/2009/USA AEI83469 2039 6449 10859, 15269,19679, 24089, 28499, 32909, 37319 105 Norovirus Hu/GI.7/TCH-060/USA/2003AEQ77282 2041 6451 10861, 15271, 19681, 24091, 28501, 32911, 37321 106Norovirus Hu/GII.1/Ascension208/2010/USA AFA55174 2042 6452 10862,15272, 19682, 24092, 28502, 32912, 37322 107 NorovirusHu/GII.13/VA173/2010/USA AFC89656 2043 6453 10863, 15273, 19683, 24093,28503, 32913, 37323 108 Norovirus Hu/GII.21/Salisbury150/2011/USAAFC89665 2046 6456 10866, 15276, 19686, 24096, 28506, 32916, 37326 109Norovirus Hu/GII.4/1997/USA AFJ04707 2049 6459 10869, 15279, 19689,24099, 28509, 32919, 37329 110 Norovirus Hu/GII.4/FarmingtonHills/2004/USA AFJ04708 2050 6460 10870, 15280, 19690, 24100, 28510,32920, 37330 111 Norovirus Hu/GII.4/Minerva/2006/USA AFJ04709 2051 646110871, 15281, 19691, 24101, 28511, 32921, 37331 112 NorovirusHu/GII.4/Ohio/71/2012/USA AFP89593 2052 6462 10872, 15282, 19692, 24102,28512, 32922, 37332 113 Norovirus Hu/GII.4/AlbertaE1065/2011/CA AFU557312068 6478 10888, 15298, 19708, 24118, 28528, 32938, 37348 114 NorovirusHu/GII.4/SG4051-09/2009/SG AFU92710 2119 6529 10939, 15349, 19759,24169, 28579, 32989, 37399 115 Norovirus Hu/GII.3/TCH-104/USA/2002AGO64038 2170 6580 10990, 15400, 19810, 24220, 28630, 33040, 37450 116Norovirus Hu/GI.6/TCH-099/USA/2003 AGT62521 2174 6584 10994, 15404,19814, 24224, 28634, 33044, 37454 117 Norovirus06-AM-I1/2006/GII.4/Yerseke/20006a AJZ77004 2228 6638 11048, 15458.19868, 24278, 28688, 33098, 37508 118 Norovirus09-BI-2/2009/GII.4/NewOrleans/2009 AJZ77015 2231 6641 11051, 15461,19871, 24281, 28691, 33101, 37511 119 Norovirus Hu/GII.4/PR328/2013/ITAAKE31861 2242 6652 11062, 15472, 19882, 24292, 28702, 33112, 37522 120Norovirus Hu/GII.P17_GII.17/PR668/2015/ITA ALD09618 2253 6663 11073,15483, 19893, 24303, 28713, 33123, 37533 121 NorovirusHu/GII.4/AlbertaSP1/2013/CA ALT54494 2274 6684 11094, 15504, 19914,24324, 28734, 33144, 37554 122 Norovirus Hu/GII.4/C00007892/2011/UKCCX28619 2280 6690 11100, 15510, 19920, 24330, 28740, 33150, 37560 123Norovirus Hu/GII.6/GZ2010-L96/Guangzhou/CHN/2011 AGC96535 2330 674011150, 15560, 19970, 24380, 28790, 33200, 37610 124 NorovirusBo/GIII.1/Aba-Z5/2002/HUN ABY67257 2341 6751 11161, 15571, 19981, 24391,28801, 33211, 37621 125 Norovirus GI.9 AHA91656 2342 6752 11162, 15572,19982, 24392, 28802, 33212, 37622 126 NorovirusHu/GII.17/CUHK-NS-670/HKG/2015 KT315718 2488 6898 11308, 15718, 20128,24538, 28948, 33358, 37768 127 NorovirusGII/Hu/SI/2015/GII.17/Ljubljana1662 KT591501 2509 6919 11329, 15739,20149, 24559, 28969, 33379, 37789 128 NorovirusHu/GII.17/CUHK-NS-647/HKG/2015 KT315706 2529 6939 11349, 15759, 20169,24579, 28989, 33399, 37809 129 Norovirus Hu/GII.21/CUHK-NS-609/HKG/2015KR921940 2540 6950 11360, 15770, 20180, 24590, 29000, 33410, 37820 130Norovirus Hu/GII.4/Melbourne6623/2016/AUS KX767083 2600 7010 11420,15830, 20240, 24650, 29060, 33470, 37880 131 NorovirusGII/Hu/JP/2016/GII.P16_GII.4_Sydney2012/OH16002 LC153121 2664 707411484, 15894, 20304, 24714, 29124, 33534, 37944 132 NorovirusHu/GII/JP/2016/GII.P16_GII.4_Sydney2012/Kawasaki194 LC175468 3557 796612376, 16786, 21196, 25606, 30016, 34426, 38836 133 Norovirus16F2149_GII.2_Guangdong_CHN_2016 KY485125 2438 6848 11258, 15668, 20078,24488, 28898, 33308, 37718 134 Norovirus Hu/GII.17/CUHK-NS-864/HKG/2016KU555841 2594 7004 11414, 15824, 20234, 24944, 29054, 33494, 37874 135Norovirus GII/Hu/ZAF/2012/GII.P4_GII.4/CapeTown/9772 KP784696 2613 702311433, 15843, 20253, 24663, 29073, 33483, 37893 136 Norovirus GII.12KP064099 4036 8446 12859, 17299, 21979, 29086, 30496, 34906, 39316 137Snow Mountain virus U70059 2441 6851 11261, 15671, 20081, 24491, 28901,33311, 37721 138 Human calicivirus strain Melksham X81879 2359 676911179, 15589, 19999, 24409, 28819, 33229, 37639

Preferably, the molar ratio of the complexed nucleic acid to the freenucleic acid is selected from a molar ratio of about 0.001:1 to about1:0.001, including a ratio of about 1:1. In a preferred embodiment, theinvention provides a composition comprising at least one artificialnucleic acid as described herein, wherein the ratio of complexed nucleicacid to free nucleic acid is selected from a range of about 5:1 (w/w) toabout 1:10 (w/w), more preferably from a range of about 4:1 (w/w) toabout 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) toabout 1:5 (w/w) or 1:3 (w/w), wherein the ratio is mast preferably about1:1 (w/w).

In one embodiment, at least one artificial nucleic acid as definedherein or any other nucleic acid comprised in the inventive(pharmaceutical) composition or vaccine can also be associated with avehicle, transfection or complexation agent for increasing thetransfection efficiency and/or the immunostimulatory properties of theat least one artificial nucleic acid or of optionally comprised furtherincluded nucleic acids.

In the context of the present invention, a cationic or polycationiccompound is preferably selected from any cationic or polycationiccompound, suitable for complexing and thereby stabilizing a nucleicacid, particularly the at least one artificial nucleic acid of theinventive composition, e.g. by associating the at least one artificialnucleic acid with the cationic or polycationic compound. Such a cationicor polycationic compound per se does not need to exhibit any adjuvantproperties, since an adjuvant property, particularly the capability ofinducing an innate immune response, is preferably created uponcomplexing the at least one artificial nucleic acid with the cationic orpolycationic compound. When complexing the at least one artificialnucleic acid with the cationic or polycationic compound, the adjuvantcomponent is formed.

Particularly preferred, cationic or polycationic peptides or proteins(preferably also as component P² in a polymeric carrier according toformula IV herein) may be selected from protamine, nucleoline, spermineor spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine,cell penetrating peptides (CPPs), chimeric CPPs, such as Transportan, orMPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derivedpeptides, oligoarginines, members of the penetratin family, e.g.Penetratin, Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, etc., antimicrobial-derived CPPs e.g.Buforin-2, Bac715-24, SynB, SynB(1), pVEC, ET-derived peptides, SAP,MAP, KALA, PpTG20, Proline-rich peptides, L-oligomers. Arginine-richpeptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine,poly-Arginine, histones, VP22 derived or analog peptides, HSV, VP22(Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, Pep-1, Calcitonin peptide(s), etc.

According to a preferred embodiment, cationic or polycationic proteinsor peptides are selected from the following proteins or peptides havingthe following total formula (III):

(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),  formula (III)

wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8, provided, that theoverall content of Xaa does not exceed 50% of all amino acids of theoligopeptide. Preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. In this contextthe disclosure of WO 2009/030481 is incorporated herewith by reference.

Further preferred cationic or polycationic compounds, which can be usedfor complexing the at least one artificial nucleic acid according to theinvention may include cationic polysaccharides, for example chitosan,polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationiclipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammoniumchloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,DOPE, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.Association or complexing the at least one artificial nucleic acid ofthe inventive composition with cationic or polycationic compoundspreferably provides adjuvant properties to the at least one artificialnucleic acid and confers a stabilizing effect to the at least oneartificial nucleic acid of the adjuvant component by complexation. Theprocedure for stabilizing the at least one artificial nucleic acid is ingeneral described in EP-A-1083232, the disclosure of which isincorporated by reference into the present invention in its entirety.Particularly preferred as cationic or polycationic compounds arecompounds selected from the group consisting of protamine, nucleoline,spermin, spermidine, oligoarginines as defined above, such as Arg₇,Arg₈, Arg₉, Arg₇, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc.

According to a preferred embodiment, the inventive composition isformulated by using the at least one artificial nucleic acid accordingto the invention and one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, the inventive composition comprisesliposomes. Liposomes are artificially-prepared vesicles, which mayprimarily be composed of a lipid bilayer and may be used as a deliveryvehicle for the administration of nutrients and pharmaceuticalformulations. Liposomes can be of different sizes such as, but notlimited to, a multilamellar vesicle (MLV) which may be hundreds ofnanometers in diameter and may contain a series of concentric bilayersseparated by narrow aqueous compartments, a small unicellular vesicle(SUV) which may be smaller than 50 nm in diameter, and a largeunilamellar vesicle (LUV) which may be between 50 and 500 nm indiameter. Liposome design may include, but is not limited to, opsoninsor ligands in order to improve the attachment of liposomes to unhealthytissue or to activate events such as, but not limited to, endocytosis.Liposomes may contain a low or a high pH in order to improve thedelivery of the inventive composition, in particular when applied as apharmaceutical composition or a vaccine as described herein.

According to a preferred embodiment, the inventive composition comprisesthe artificial nucleic acid as described herein and a polymeric carrier.A polymeric carrier used according to the invention might be a polymericcarrier formed by disulfide-crosslinked cationic components. Thedisulfide-crosslinked cationic components may be the same or differentfrom each other. The polymeric carrier can also contain furthercomponents. It is also particularly preferred that the polymeric carrierused in the composition according to the present invention comprisesmixtures of cationic peptides, proteins or polymers and optionallyfurther components as defined herein, which are crosslinked by disulfidebonds as described herein. In this context, the disclosure of WO2012/013326 is incorporated herewith by reference.

In this context, the cationic components, which form basis for thepolymeric carrier by disulfide-crosslinkage, are typically selected fromany suitable cationic or polycationic peptide, protein or polymersuitable for this purpose, particular any cationic or polycationicpeptide, protein or polymer capable to complex the at least oneartificial nucleic acid as defined herein or a further nucleic acidcomprised in the composition, and thereby preferably condensing the mRNAor the nucleic acid. The cationic or polycationic peptide, protein orpolymer, is preferably a linear molecule, however, branched cationic orpolycationic peptides, proteins or polymers may also be used.

Every disulfide-crosslinking cationic or polycationic protein, peptideor polymer of the polymeric carrier, which may be used to complex the atleast one artificial nucleic acid or any further nucleic acid comprisedin the inventive (pharmaceutical) composition or vaccine contains atleast one —SH moiety, most preferably at least one cysteine residue orany further chemical group exhibiting an —SH moiety, capable to form adisulfide linkage upon condensation with at least one further cationicor polycationic protein, peptide or polymer as cationic component of thepolymeric carrier as mentioned herein.

As defined above, the polymeric carrier, which may be used to complexthe at least one artificial nucleic acid or any further nucleic acidcomprised in the inventive (pharmaceutical) composition or vaccine maybe formed by disulfide-crosslinked cationic (or polycationic)components. Preferably, such cationic or polycationic peptides orproteins or polymers of the polymeric carrier, which comprise or areadditionally modified to comprise at least one —SH moiety, are selectedfrom, proteins, peptides and polymers as defined above for complexationagent.

In a further particular embodiment, the polymeric carrier which may beused to complex the at least one artificial nucleic acid or any furthernucleic acid comprised in the inventive (pharmaceutical) composition orvaccine may be selected from a polymeric carrier molecule according togeneric formula (IV):

L-P¹—S-[S—P²—S]_(n)—S—P³-L  formula (IV)

wherein,

-   P¹ and P³ are different or identical to each other and represent a    linear or branched hydrophilic polymer chain, each P¹ and P³    exhibiting at least one —SH-moiety, capable to form a disulfide    linkage upon condensation with component P², or alternatively with    (AA), (AA)_(x), or [(AA)_(x)]_(z) if such components are used as a    linker between P¹ and P² or P³ and P²) and/or with further    components (e.g. (AA), (AA)_(x), [(AA)_(x)]_(z), or L), the linear    or branched hydrophilic polymer chain selected independent from each    other from polyethylene glycol (PEG),    poly-N-(2-hydroxypropyl)methacrylamide,    poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl    L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),    hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the    hydrophilic polymer chain exhibits a molecular weight of about 1 kDa    to about 100 kDa, preferably of about 2 kDa to about 25 kDa: or more    preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to about    25 kDa or 5 kDa to about 10 kDa;-   P² is a cationic or polycationic peptide or protein, e.g. as defined    above for the polymeric carrier formed by disulfide-crosslinked    cationic components, and preferably having a length of about 3 to    about 100 amino acids, more preferably having a length of about 3 to    about 50 amino acids, even more preferably having a length of about    3 to about 25 amino acids, e.g. a length of about 3 to 10, 5 to 15,    10 to 20 or 15 to 25 amino acids, more preferably a length of about    5 to about 20 and even more preferably a length of about 10 to about    20: or    -   is a cationic or polycationic polymer, e.g. as defined above for        the polymeric carrier formed by disulfide-crosslinked cationic        components, typically having a molecular weight of about 0.5 kDa        to about 30 kDa, including a molecular weight of about 1 kDa to        about 20 kDa, even more preferably of about 1.5 kDa to about 10        kDa, or having a molecular weight of about 0.5 kDa to about 100        kDa, including a molecular weight of about 10 kDa to about 50        kDa, even more preferably of about 10 kDa to about 30 kDa;    -   each P² exhibiting at least two —SH-moieties, capable to form a        disulfide linkage upon condensation with further components P²        or component(s) P′ and/or P³ or alternatively with further        components (e.g. (AA), (AA)_(x), or [(AA)_(x)]_(z));-   —S—S— is a (reversible) disulfide bond (the brackets are omitted for    better readability), wherein S preferably represents sulphur or a    —SH carrying moiety, which has formed a (reversible) disulfide bond.    The (reversible) disulfide band is preferably formed by condensation    of —SH-moieties of either components P¹ and P², P² and P², or P² and    P³, or optionally of further components as defined herein (e.g. L,    (AA), (AA)_(x), [(AA)_(x)]_(z), etc); The —SH-moiety may be part of    the structure of these components or added by a modification as    defined below;-   L is an optional ligand, which may be present or not, and may be    selected independent from the other from ROD, Transferrin, Folate, a    signal peptide or signal sequence, a localization signal or    sequence, a nuclear localization signal or sequence (NLS), an    antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand    of a receptor (e.g. cytokines, hormones, growth factors etc), small    molecules (e.g. carbohydrates like mannose or galactose or synthetic    ligands), small molecule agonists, inhibitors or antagonists of    receptors (e.g. RGD peptidomimetic analogues), or any further    protein as defined herein, etc.;-   n is an integer, typically selected from a range of about 1 to 50,    preferably from a range of about 1, 2 or 3 to 30, more preferably    from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1,    2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or    a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of    about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a    range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of    about 6 to 11 or 7 to 10. Most preferably, n is in a range of about    1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3,    or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of    about 1, 2, or 3 to 7.

In this context, the disclosure of WO 2011/026641 is incorporatedherewith by reference. Each of hydrophilic polymers P¹ and P³ typicallyexhibits at least one —SH-moiety, wherein the at least one —SH-moiety iscapable to form a disulfide linkage upon reaction with component P² orwith component (AA) or (AA)_(x), if used as linker between P¹ and P² orP³ and P² as defined below and optionally with a further component, e.g.L and/or (AA) or (AA)_(x), e.g. if two or more —SH-moieties arecontained. The following subformulae “P¹—S—S—P²” and “P²—S—S—P³” withingeneric formula (IV) above (the brackets are omitted for betterreadability), wherein any of S, P¹ and P³ are as defined herein,typically represent a situation, wherein one-SH-moiety of hydrophilicpolymers P¹ and P³ was condensed with one —SH-moiety of component P² ofgeneric formula (IV) above, wherein both sulphurs of these —SH-moietiesform a disulfide bond —S—S— as defined herein in formula (IV). These—SH-moieties are typically provided by each of the hydrophilic polymersP¹ and P³, e.g. via an internal cysteine or any further (modified) aminoacid or compound which carries a —SH moiety. Accordingly, thesubformulae “P¹—S—S—P²” and “P²—S—S—P³” may also be written as“P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the —SH— moiety is provided by acysteine, wherein the term Cys-Cys represents two cysteines coupled viaa disulfide bond, not via a peptide bond. In this case, the term “—S—S—”in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as“-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent apeptide bond but a linkage of two cysteines via their —SH-moieties toform a disulfide bond. Accordingly, the term “-Cys-Cys-” also may beunderstood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specificcase S indicates the sulphur of the —SH-moiety of cysteine. Likewise,the terms “—S-Cys” and “-Cys-S” indicate a disulfide bond between a SHcontaining moiety and a cysteine, which may also be written as“—S—(S-Cys)” and “-(Cys-S)—S”. Alternatively, the hydrophilic polymersP¹ and P³ may be modified with a —SH moiety, preferably via a chemicalreaction with a compound carrying a —SH moiety, such that each of thehydrophilic polymers P¹ and P³ carries at least one such —SH moiety.Such a compound carrying a —SH moiety may be e.g. an (additional)cysteine or any further (modified) amino acid, which carries a —SHmoiety. Such a compound may also be any non-amino compound or moiety,which contains or allows to introduce a —SH moiety into hydrophilicpolymers P¹ and P³ as defined herein. Such non-amino compounds may beattached to the hydrophilic polymers P¹ and P³ of formula (IV) of thepolymeric carrier according to the present invention via chemicalreactions or binding of compounds, e.g. by binding of a 3-thio propionicacid or thioimolane, by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc), by Michael addition (e.g maleinimidemoieties, α,β-unsatured carbonyls, etc), by click chemistry (e.g. azidesor alkines), by alkene/alkine methatesis (e.g. alkenes or alkines),imine or hydrozone formation (aldehydes or ketons, hydrazine,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components. Aparticularly preferred PEG derivate in this context isalpha-methoxy-omega-mercapto polyethylene glycol). In each case, theSH-moiety, e.g. of a cysteine or of any further (modified) amino acid orcompound, may be present at the terminal ends or internally at anyposition of hydrophilic polymers P¹ and P³. As defined herein, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one SH-moietypreferably at one terminal end, but may also contain two or even moreSH-moieties, which may be used to additionally attach further componentsas defined herein, preferably further functional peptides or proteinse.g. a ligand, an amino acid component (AA) or (AA)_(x), antibodies,cell penetrating peptides or enhancer peptides (e.g. TAT, KALA), etc.

The complexed artificial nucleic acid in the inventive (pharmaceutical)composition or vaccine, is preferably prepared according to a first stepby complexing the at least one artificial with a cationic orpolycationic compound and/or with a polymeric carrier, preferably asdefined herein, in a specific ratio to form a stable complex. In thiscontext, it is highly preferable, that no free cationic or polycationiccompound or polymeric carrier or only a negligibly small amount thereofremains in the component of the complexed artificial nucleic acid aftercomplexing the artificial nucleic acid. Accordingly, the ratio of the atleast one artificial nucleic acid and the cationic or polycationiccompound and/or the polymeric carrier in the component of the complexedat least one artificial nucleic acid is typically selected in a rangethat the at least one artificial nucleic acid is entirely complexed andno free cationic or polycationic compound or polymeric carrier or only anegligibly small amount thereof remains in the composition.

The inventive composition comprising at least one artificial nucleicacid according to the invention may be provided in liquid and or in dry(e.g. lyophylized) form. In a preferred embodiment, the inventiveartificial nucleic acid or the inventive composition is provided inlyophilized form. The inventive artificial nucleic acid and theinventive composition thus provide a possibility to store (irrespectiveof the ambient temperature and also without cooling) an artificialnucleic acid and a composition suitable for vaccination againstNorovirus and related diseases or disorders. Preferably, the at leastone lyophilized artificial nucleic acid is reconstituted in a suitablebuffer, advantageously based on an aqueous carrier, e.g. Ringer-Lactatesolution, prior to use, such as administration to a subject.

In one embodiment, the composition according to the invention

-   -   a) comprises a plurality or more than one of the mRNA sequences        as defined in the invention;    -   or    -   b) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,        78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,        94, 95, 96, 97, 98, 99, 100 or more artificial nucleic acids as        defined in the invention, wherein each of the at least 2, 3, 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,        38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,        54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,        70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,        86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or        more artificial nucleic acids comprises at least one coding        region encoding at least one polypeptide comprising a Norovirus        protein as defined in the invention, and/or a fragment or a        variant of any one of these proteins, wherein each coding region        preferably encodes a different Norovirus protein, more        preferably each coding region encodes a capsid protein,        preferably VP1 of a different Norovirus.

In another embodiment, the composition according to the inventioncomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more artificialnucleic acids of the invention, wherein each of the at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 or more artificial nucleic acids comprises at least onecoding region encoding at least one polypeptide comprising at least twodifferent Norovirus proteins, preferably VP1 and VP2, as defined in theinvention, and/or a fragment or a variant of any one of these proteins,and/or wherein each of the mRNA sequences encodes at least one differentantigenic peptide or protein derived from proteins of the sameNorovirus.

In a further embodiment, the composition according to the inventioncomprises mRNA sequences, wherein each of the mRNA sequences encodes atleast one different antigenic peptide or protein derived from differentproteins of the same Norovirus.

In a further embodiment, the composition according to the inventioncomprises mRNA sequences, wherein each of the mRNA sequences encodes atleast one different antigenic peptide or protein derived from differentproteins of different Noroviruses.

In a further aspect, the invention concerns a vaccine comprising theartificial nucleic acid as described herein or the inventive compositioncomprising at least one artificial nucleic acid according to theinvention. Therein, the at least one artificial nucleic acid preferablyelicits an adaptive immune response upon administration to a subject.

In a preferred embodiment, the inventive vaccine comprises theartificial nucleic acid as described herein or the inventive compositioncomprising at least one artificial nucleic acid according to theinvention and a pharmaceutically acceptable carrier. Accordingly, theinventive vaccine is based on the same components as the inventivecomposition comprising at least one artificial nucleic acid according tothe invention as defined above. Insofar, it may be referred to the abovedisclosure defining the inventive composition.

In one embodiment, the composition of the invention comprises aplurality or more than one of the mRNA sequences of the invention.

In one embodiment, the composition of the invention comprises aplurality or more than one of the mRNA sequences of the invention,wherein each of the mRNA sequences encodes at least one differentantigenic peptide or protein derived from proteins of the sameNorovirus.

In one embodiment, the composition of the invention comprises aplurality or more than one of the mRNA sequences of the invention,wherein each of the mRNA sequences encodes at least one differentantigenic peptide or protein derived from different proteins of the sameNorovirus.

In another embodiment, the composition of the invention comprises aplurality or more than one of the mRNA sequences of the invention,wherein each of the mRNA sequences encodes at least one differentantigenic peptide or protein derived from different proteins ofdifferent Noroviruses.

In one embodiment, the vaccine of the invention is multivalent andcomprises

-   -   (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more artificial        nucleic acids of the invention; or    -   (ii) at least 10, 15, 20 or 50 artificial nucleic acids of the        invention; or    -   (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificial        nucleic acids of the invention.

In a further embodiment of the invention, the artificial nucleic acidsof the vaccine of the invention

-   -   (i) are derived from a single GI Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        different GI Noroviruses; or    -   (ii) are derived from a single GII Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, BE, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        different GII Noroviruses; or    -   (iii) are derived from a single G111 Norovirus or from 2, 3, 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,        38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,        54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,        70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,        86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or        more different GIII Noroviruses; or    -   (iv) are derived from a single GIV Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        different GIV Noroviruses; or    -   (v) are derived from a single GV Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        different GV Noroviruses; or    -   (vi) are derived from a single GI Norovirus and additionally        from a single GII Norovirus, GIII Norovirus, GIV Norovirus        and/or GV Norovirus: or    -   (vii) are derived from a single GI Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76.77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        different GI Noroviruses and additionally from a single G11,        G111, GIV and/or GV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,        26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,        42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,        58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,        74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,        90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more GII, GIII,        GIV and/or GV Noroviruses.

In another embodiment of the invention, the artificial nucleic acids ofthe vaccine of the invention

-   -   (i) are derived from a single GI.1 Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,        55, 56, 57, 58, 59, BO, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,        71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more        different GI.1 Noroviruses: or    -   (ii) are derived from a single 011.4 Norovirus or from 2, 3, 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,        38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,        54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,        70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,        86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or        more different GII.4 Noroviruses; or    -   (iii) are derived from a single GI.1 Norovirus and additionally        from a single GII.4 Norovirus; or    -   (iv) are derived from a single GI.1 Norovirus or from 2, 3, 4,        5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,        38, 39, 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50, 51, 52, 53,        54, 55, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,        70, 71, 72, 73, 74, 75, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85,        86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 97, 98, 99, 100 or        more different GI.1 Noroviruses and additionally from a single        GII.4 Norovirus or from 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 27, 28, 29,        30, 31, 32, 33, 34, 35, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45,        45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 75, 77,        78, 79, 80, 81, 82, 83, 84, 85, 85, 87, 88, 89, 90, 91, 92, 93,        94, 95, 95, 97, 98, 99, 100 or more GII.4 Noroviruses.

As with the composition according to the present invention, the entitiesof the vaccine may be provided in liquid and or in dry (e.g.lyophylized) form. They may contain further components, in particularfurther components allowing for its pharmaceutical use. The inventivevaccine or the inventive composition may, e.g., additionally contain apharmaceutically acceptable carrier and/or further auxiliary substancesand additives and/or adjuvants.

The inventive vaccine or composition typically comprises a safe andeffective amount of the inventive artificial nucleic acid as definedherein. As used herein, “safe and effective amount” means an amount ofthe artificial nucleic acid of the composition or the vaccine as definedabove, that is sufficient to significantly induce an immune responseagainst a Norovirus protein. At the same time, however, a “safe andeffective amount” is small enough to avoid serious side effects that isto say to permit a sensible relationship between advantage and risk. Thedetermination of these limits typically lies within the scope ofsensible medical judgment. In relation to the inventive vaccine orcomposition, the expression “safe and effective amount” preferably meansan amount of the artificial nucleic acid that is suitable forstimulating the adaptive immune system in such a manner that noexcessive or damaging immune reactions are achieved but, preferably,also no such immune reactions below a measurable level. Such a “safe andeffective amount” of the artificial nucleic acid of the composition orvaccine as defined above may furthermore be selected in dependence ofthe type of artificial nucleic acid, e.g. monocistronic, bi- or evenmulticistronic mRNA, since a bi- or even multicistronic mRNA may lead toa significantly higher expression of the encoded polypeptide(s) than useof an equal amount of a monocistronic mRNA. A “safe and effectiveamount” of the artificial nucleic acid of the composition or vaccine asdefined above may furthermore vary in connection with the particularobjective of the treatment and also with the age and physical conditionof the patient to be treated, and similar factors, within the knowledgeand experience of the accompanying doctor. The vaccine or compositionaccording to the invention can be used according to the invention forhuman and also for veterinary medical purposes, as a pharmaceuticalcomposition or as a vaccine.

In a preferred embodiment, the artificial nucleic acid of thecomposition, vaccine or kit of parts according to the invention isprovided in lyophilized form. Preferably, the lyophilized artificialnucleic acid is reconstituted in a suitable buffer, advantageously basedon an aqueous carrier, prior to administration, e.g. Ringer-Lactatesolution, which is preferred, Ringer solution, a phosphate buffersolution.

According to a preferred embodiment, the buffer suitable for injectionmay be used as a carrier in the inventive vaccine or composition or forresuspending the inventive vaccine or the inventive composition. Such abuffer suitable for injection may contain salts selected from sodiumchloride (NaCl), calcium chloride (CaCl₂) and optionally potassiumchloride (KCl), wherein further anions may be present additional to thechlorides. CaCl₂ can also be replaced by another salt like KCl.Typically, the salts in the injection buffer are present in aconcentration of at least 50 mM sodium chloride (NaCl), at least 3 mMpotassium chloride (KCl) and at least 0.01 mM calcium chloride (CaCl₂).The injection buffer may be hypertonic, isotonic or hypotonic withreference to the specific reference medium, i.e. the buffer may have ahigher, identical or lower salt content with reference to the specificreference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due toosmosis or other concentration effects. Reference media are e.g. in “invivo” methods occurring liquids such as blood, lymph, cytosolic liquids,or other body liquids, or e.g. liquids, which may be used as referencemedia in “in vitro” methods, such as common buffers or liquids. Suchcommon buffers or liquids are known to a skilled person. Ringer-Lactatesolution is particularly preferred as a liquid basis.

The choice of a pharmaceutically acceptable carrier is determined, inprinciple, by the manner, in which the inventive vaccine or theinventive composition is administered. The inventive vaccine orcomposition can be administered, for example, systemically or locally.Routes for systemic administration in general include, for example,transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,the inventive vaccine or the inventive composition may be administeredby an intradermal, subcutaneous, or intramuscular route, preferably byinjection, which may be needle-free and/or needle injection.Compositions/vaccines are therefore preferably formulated in liquid orsolid form. The suitable amount of the inventive vaccine or compositionto be administered can be determined by routine experiments with animalmodels. Such models include, without implying any limitation, rabbit,sheep, mouse, rat, dog and non-human primate models. Preferred unit doseforms for injection include sterile solutions of water, physiologicalsaline or mixtures thereof. The pH of such solutions should be adjustedto about 7.4. Suitable carriers for injection include hydrogels, devicesfor controlled or delayed release, polylactic acid and collagenmatrices. Suitable pharmaceutically acceptable carriers for topicalapplication include those which are suitable for use in lotions, creams,gels and the like. If the inventive vaccine is to be administeredperorally, tablets, capsules and the like are the preferred unit doseform. The pharmaceutically acceptable carriers for the preparation ofunit dose forms which can be used for oral administration are well knownin the prior art. The choice thereof will depend on secondaryconsiderations such as taste, costs and storability, which are notcritical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

Adjuvants:

According to another embodiment, the inventive (pharmaceutical)composition or the inventive vaccine may comprise an adjuvant. Anadjuvant may be used, for example, in order to enhance theimmunostimulatory properties of the vaccine or composition. In thiscontext, an adjuvant may be understood as any compound, which issuitable to support administration and delivery of the vaccine orcomposition according to the invention. Furthermore, such an adjuvantmay, without being bound thereto, initiate or increase an immuneresponse of the innate immune system, i.e. a non-specific immuneresponse. In other words, when administered, the vaccine or compositionaccording to the invention typically initiates an adaptive immuneresponse due to the at least one polypeptide encoded by the artificialnucleic acid contained in the inventive vaccine or composition.Additionally, the vaccine or composition according to the invention maygenerate an (supportive) innate immune response due to addition of anadjuvant as defined herein to the vaccine or composition according tothe invention.

In one embodiment, the adjuvant is selected from the group consistingof:

cationic or polycationic compounds, comprising cationic or polycationicpeptides or proteins, including protamine, nucleoline, spermin orspermidine, poly-L-lysine (PLL), poly-arginine, basic polypeptides, cellpenetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analogpeptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transductiondomains (PTDs, PpT620, proline-rich peptides, arginine-rich peptides,lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitoninpeptide(s), Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine,spermine, spermidine, or histones, cationic polysaccharides, includingchitosan, polybrene, cationic polymers, including polyethyleneimine(PEI), cationic lipids, including DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, OC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonia)prapane, DC-6-14:D,D-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, including modifiedpolyaminoacids, including β-aminoacid-polymers or reversed polyamides,modified polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridiniumbromide)), modified acrylates, including pDMAEMA(poly(dimethylaminoethyl methylacrylate)), modified Amidoaminesincluding pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE),including diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, dendrimers, includingpolypropylamine dendrimers or pAMAM based dendrimers, polyimine(s),including PEI: poly(ethyleneimine), poly(propyleneimine),polyallylamine, sugar backbone based polymers, including cyclodextrinbased polymers, dextran based polymers, chitosan, etc., silan backbonebased polymers, such as PMOXA-PDMS copolymers, etc., block polymersconsisting of a combination of one or more cationic blocks selected froma cationic polymer as mentioned before, and of one or more hydrophilic-or hydrophobic blocks (e.g polyethyleneglycole);orcationic or polycationic proteins or peptides, selected from thefollowing proteins or peptides having the following total formula (III):(Arg)l; (Lys)m; (His)n; (Orn)o; (Xaa)x, wherein l+m+n+o+x=8-15, and l,m, n or o independently of each other may be any number selected from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that theoverall content of Arg, Lys, His and Orn represents at least 50% of allamino acids of the oligopeptide; and Xaa may be any amino acid selectedfrom native (=naturally occurring) or non-native amino acids except fromArg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3or 4, provided, that the overall content of Xaa does not exceed 50% ofall amino acids of the oligopeptide; ornucleic acids having the formula (V): GlXmGn, wherein: G is guanosine,uracil or an analogue of guanosine or uracil; X is guanosine, uracil,adenosine, thymidine, cytosine or an analogue of the above-mentionednucleotides; I is an integer from 1 to 40, wherein, when I=1 G isguanosine or an analogue thereof, when I>1 at least 50% of thenucleotides are guanosine or an analogue thereof; m is an integer and isat least 3; wherein when m=3 X is uracil or an analogue thereof, whenm>3 at least 3 successive uracils or analogues of uracil occur; n is aninteger from 1 to 40, wherein when n=1 G is guanosine or an analoguethereof, when n>1 at least 50% of the nucleotides are guanosine or ananalogue thereof;ornucleic acids having the formula (VI): CIXmCn, wherein: C is cytosine,uracil or an analogue of cytosine or uracil; X is guanosine, uracil,adenosine, thymidine, cytosine or an analogue of the above-mentionednucleotides; I is an integer from 1 to 40, wherein when I=1 C iscytosine or an analogue thereof, when I>1 at least 50% of thenucleotides are cytosine or an analogue thereof; m is an integer and isat least 3; wherein when m=3 X is uracil or an analogue thereof, whenm>3 at least 3 successive uracils or analogues of uracil occur; n is aninteger from 1 to 40, wherein when n=1 Cis cytosine or an analoguethereof, when n>1 at least 50% of the nucleotides are cytosine or ananalogue thereof;oradjuvants selected from the group consisting of:TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminiumhydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucansfrom algae; algammulin; aluminium hydroxide gel (alum); highlyprotein-adsorbing aluminium hydroxide gel; low viscosity aluminiumhydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%),Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™(propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DOA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin: Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP),ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline saltcomplex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-O-isoglutamine);imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon-gamma; interleukin-(beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-0-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L112™; PMMA(polymethyl methacrylate); PODDS™ (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™(Q5-21); Quil-A (Quil-A saponin); S-28403(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendaiproteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitantrioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85);squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIDRAL; plant derived adjuvants, including Q521, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin; microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR1-10, ligands of murineTLR1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine,IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPIanchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusionprotein, cdiGMP; and adjuvants suitable as antagonists including CGRPneuropeptide.

Particularly preferred are aluminium salts, such as aluminium phosphate(AlPO₄) or aluminium hydroxide (Al(OH)₃) and adjuvant compounds basedthereon. More preferably, an aluminium salt, such as AlPO₄ (e.g.Adju-Phos) may be used in combination with the inventive artificialnucleic acid in its free form or with the inventive artificial nucleicacid complexed with a cationic or polycationic compound as describedherein. Most preferably, an aluminium salt, such as AlPO₄ (e.g.Adju-Phos) may be used as adjuvant in combination with the inventiveartificial nucleic acid in its free form.

Suitable adjuvants may also be selected from cationic or polycationiccompounds, preferably as described herein, wherein the adjuvant ispreferably prepared upon complexing the at least one artificial nucleicacid of the inventive composition or vaccine with the cationic orpolycationic compound. Association or complexing the artificial nucleicacid with cationic or polycationic compounds as defined hereinpreferably provides adjuvant properties and confers a stabilizing effectto the artificial nucleic acid.

The ratio of the artificial nucleic acid to the cationic or polycationiccompound in the adjuvant component may be calculated on the basis of thenitrogen/phosphate ratio (N/P-ratio) of the entire artificial nucleicacid complex, i.e. the ratio of positively charged (nitrogen) atoms ofthe cationic or polycationic compound to the negatively chargedphosphate atoms of the nucleic acids. For example, 1 μg RNA typicallycontains about 3 nmol phosphate residues, provided the RNA exhibits astatistical distribution of bases. Additionally, 1 μg peptide typicallycontains about x nmol nitrogen residues, dependent on the molecularweight and the number of basic amino acids. When exemplarily calculatedfor (Arg)s (molecular weight 1424 g/mol, 9 nitrogen atoms), 1 μg (Arg)scontains about 700 pmol (Arg)₉ and thus 700×9=6300 pmol basic aminoacids=6.3 nmol nitrogen atoms. For a mass ratio of about 1:1 RNA/(Arg)9an N/P ratio of about 2 can be calculated. When exemplarily calculatedfor protamine (molecular weight about 4250 g/mol, 21 nitrogen atoms,when protamine from salmon is used) with a mass ratio of about 2:1 with2 μg RNA, 6 nmol phosphate are to be calculated for the RNA; 1 μgprotamine contains about 235 pmol protamine molecules and thus235×21=4935 pmol basic nitrogen atoms=4.9 nmol nitrogen atoms. For amass ratio of about 2:1 RNA/protamine an N/P ratio of about 0.81 can becalculated. For a mass ratio of about 8:1 RNA/protamine an N/P ratio ofabout 0.2 can be calculated. In the context of the present invention, anN/P-ratio is preferably in the range of about 0.1-10, preferably in arange of about 0.3-4 and most preferably in a range of about 0.5-2 or0.7-2 regarding the ratio of nucleic acid:peptide in the complex, andmost preferably in the range of about 0.7-1.5.

In a preferred embodiment, the inventive vaccine or the inventivecomposition is obtained in two separate steps in order to obtain both,an efficient immunostimulatory effect and efficient translation of theartificial nucleic acid according to the invention. Therein, a so called“adjuvant component” is prepared by complexing—in a first step—a nucleicacid, preferably an RNA, of the adjuvant component with a cationic orpolycationic compound in a specific ratio to form a stable complex. Inthis context, it is important, that no free cationic or polycationiccompound or only a neglibly small amount remains in the adjuvantcomponent after complexing the nucleic acid. Accordingly, the ratio ofthe nucleic acid, preferably an RNA, and the cationic or polycationiccompound in the adjuvant component is typically selected in a range thatthe artificial nucleic acid is entirely complexed and no free cationicor polycationic compound or only a neglectably small amount remains inthe composition. Preferably the ratio of the adjuvant component, i.e.the ratio of the artificial nucleic acid to the cationic or polycationiccompound is selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w).

According to a preferred embodiment, the artificial nucleic acid,preferably an mRNA, is added in a second step to the complexed nucleicacid, preferably an RNA, of the adjuvant component in order to form the(immunostimulatory) composition of the invention. Therein, theartificial nucleic acid is added as free nucleic acid, i.e. nucleicacid, which is not complexed by other compounds. Prior to addition, thefree artificial nucleic acid is not complexed and will preferably notundergo any detectable or significant complexation reaction upon theaddition of the adjuvant component. This is due to the strong binding ofthe cationic or polycationic compound to the above described artificialnucleic acid in the adjuvant component. In other words, when theartificial nucleic acid according to the invention, is added to the“adjuvant component”, preferably no free or substantially no freecationic or polycationic compound is present, which may form a complexwith the free artificial nucleic acid. Accordingly, an efficienttranslation of the free artificial nucleic acid of the inventive vaccineor composition is possible in vivo. Therein, the free artificial nucleicacid may occur, for example, as a mono-, di-, or multicistronic nucleicacid, i.e. an artificial nucleic acid which carries the coding sequencesof one or more polypeptides. Such coding sequences in a di-, or evenmulticistronic nucleic acid may be separated by at least one IRESsequence, e.g. as defined herein.

In a particularly preferred embodiment, the free artificial nucleicacid, which is comprised in the inventive vaccine or composition, may beidentical or different to the RNA of the adjuvant component of theinventive composition, depending on the specific requirements oftherapy. Even more preferably, the artificial nucleic acid, preferablyan mRNA, which is comprised in the inventive vaccine or composition, isidentical to the RNA of the adjuvant component of the inventive vaccineor composition.

In a particularly preferred embodiment, the composition comprises theartificial nucleic acid, preferably an mRNA, wherein said artificialnucleic acid is present in the composition partially as free nucleicacid and partially as complexed nucleic acid. Preferably, the artificialnucleic acid, preferably an mRNA, is complexed as described above andthe same artificial nucleic acid is then added as free nucleic acid,wherein preferably the compound, which is used for complexing theartificial nucleic acid is not present in free form in the compositionat the moment of addition of the free nucleic acid component.

The ratio of the first component (i.e. the adjuvant component comprisingor consisting of artificial nucleic acid complexed with a cationic orpolycationic compound) and the second component (i.e. the free nucleicacid) may be selected in the inventive composition according to thespecific requirements of a particular therapy. Typically, the ratio ofthe nucleic acid, preferably an RNA, in the adjuvant component and theat least one free artificial nucleic acid, preferably an mRNA,(artificial nucleic acid, preferably mRNA in the adjuvant component:freeRNA) of the inventive composition is selected such that a significantstimulation of the innate immune system is elicited due to the adjuvantcomponent. In parallel, the ratio is selected such that a significantamount of the at least one free artificial nucleic acid, preferably anmRNA, can be provided in vivo leading to an efficient translation andconcentration of the expressed protein in vivo, e.g. the at least oneencoded polypeptide as defined herein. Preferably, the ratio of the mRNAin the adjuvant component:free mRNA in the inventive composition isselected from a range of about 5:1 (w/w) to about 1:10 (w/w), morepreferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even morepreferably from a range of about 3:1(w/w) to about 1:5 (w/w) or 1:3(w/w), and most preferably the ratio of mRNA in the adjuvantcomponent:free mRNA in the inventive composition is selected from aratio of about 1:1 (w/w).

Additionally or alternatively, the ratio of the first component (i.e.the adjuvant component comprising or consisting of artificial nucleicacid complexed with a cationic or polycationic compound) and the secondcomponent (i.e. free artificial nucleic acid) may be calculated on thebasis of the nitrogen/phosphate ratio (N/P-ratio) of the entire mRNAcomplex. In the context of the present invention, an N/P-ratio ispreferably in the range of about 0.1-10, preferably in a range of about0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regardingthe ratio of mRNA:peptide in the complex, and most preferably in therange of about 0.7-1.5.

Additionally or alternatively, the ratio of the first component (i.e.the adjuvant component comprising or consisting of artificial nucleicacid, preferably mRNA, complexed with a cationic or polycationiccompound) and the second component (i.e. free artificial nucleic acid,preferably mRNA) may also be selected in the inventive composition onthe basis of the molar ratio of both nucleic acids to each other, i.e.the nucleic acid of the adjuvant component, being complexed with acationic or polycationic compound and the free nucleic acid of thesecond component. Typically, the molar ratio of the nucleic acid of theadjuvant component to the free nucleic acid of the second component maybe selected such, that the molar ratio suffices the above (w/w) and/orN/P-definitions. More preferably, the molar ratio of the nucleic acid,preferably an mRNA, of the adjuvant component to the free nucleic acid,preferably an mRNA, of the second component may be selected e.g. from amolar ratio of about 0.001:1, 0.01:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1,0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5,1:0.4, 1:0.3, 1:0.2, 1:0.1, 1:0.01, 1:0.001, etc. or from any rangeformed by any two of the above values, e.g. a range selected from about0.001:1 to 1:0.001, including a range of about 0.01:1 to 1:0.001, 0.1:1to 1:0.001, 0.2:1 to 1:0.001, 0.3:1 to 1:0.001, 0.4:1 to 1:0.001, 0.5:1to 1:0.001, 0.6:1 to 1:0.001, 0.7:1 to 1:0.001, 0.8:1 to 1:0.001, 0.9:1to 1:0.001, 1:1 to 1:0.001, 1:0.9 to 1:0.001, 1:0.8 to 1:0.001, 1:0.7 to1:0.001, 1:0.6 to 1:0.001, 1:0.5 to 1:0.001, 1:0.4 to 1:0.001, 1:0.3 to1:0.001, 1:0.2 to 1:0.001, 1:0.1 to 1:0.001, 1:0.01 to 1:0.001, or arange of about 0.01:1 to 1:0.01, 0.1:1 to 1:0.01, 0.2:1 to 1:0.01, 0.3:1to 1:0.01, 0.4:1 to 1:0.01, 0.5:1 to 1:0.01, 0.6:1 to 1:0.01, 0.7:1 to1:0.01, 0.8:1 to 1:0.01, 0.9:1 to 1:0.01, 1:1 to 1:0.01, 1:0.9 to1:0.01, 1:0.8 to 1:0.01, 1:0.7 to 1:0.01, 1:0.6 to 1:0.01, 1:0.5 to1:0.01, 1:0.4 to 1:0.01, 1:0.3 to 1:0.01, 1:0.2 to 1:0.01, 1:0.1 to1:0.01, 1:0.01 to 1:0.01, or including a range of about 0.001:1 to1:0.01, 0.001:1 to 1:0.1, 0.001:1 to 1:0.2, 0.001:1 to 1:0.3, 0.001:1 to1:0.4, 0.001:1 to 1:0.5, 0.001:1 to 1:0.6, 0.001:1 to 1:0.7, 0.001:1 to1:0.8, 0.001:1 to 1:0.9, 0.001:1 to 1:1, 0.001 to 0.9:1, 0.001 to 0.8:1,0.001 to 0.7:1, 0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to 0.4:1, 0.001 to0.3:1, 0.001 to 0.2:1, 0.001 to 0.1:1, or a range of about 0.01:1 to1:0.01, 0.01:1 to 1:0.1, 0.01:1 to 1:0.2, 0.01:1 to 1:0.3, 0.01:1 to1:0.4, 0.01:1 to 1:0.5, 0.01:1 to 1:0.6, 0.01:1 to 1:0.7, 0.01:1 to1:0.8, 0.01:1 to 1:0.9, 0.01:1 to 1:1, 0.001 to 0.9:1, 0.001 to 0.8:1,0.001 to 0.7:1, 0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to 0.4:1, 0.001 to0.3:1, 0.001 to 0.2:1, 0.001 to 0.1:1, etc.

Even more preferably, the molar ratio of the artificial nucleic acid,preferably an mRNA, of the adjuvant component to the free nucleic acid,preferably an mRNA, of the second component may be selected e.g. from arange of about 0.01:1 to 1:0.01. Most preferably, the molar ratio of thenucleic acid of the adjuvant component to the free nucleic acid of thesecond component may be selected e.g. from a molar ratio of about 1:1.Any of the above definitions with regard to (w/w) and/or N/P ratio mayalso apply.

Suitable adjuvants may furthermore be selected from nucleic acids havingthe formula (V): G_(l)X_(m)G_(n), wherein: G is guanosine, uracil or ananalogue of guanosine or uracil; X is guanosine, uracil, adenosine,thymidine, cytosine or an analogue of the above-mentioned nucleotides; Iis an integer from 1 to 40, wherein when I=1 G is guanosine or ananalogue thereof, when I>1 at least 50% of the nucleotides are guanosineor an analogue thereof; m is an integer and is at least 3; wherein whenm=3 X is uracil or an analogue thereof, when m>3 at least 3 successiveuracils or analogues of uracil occur; n is an integer from 1 to 40,wherein when n=1 G is guanosine or an analogue thereof, when n>1 atleast 50% of the nucleotides are guanosine or an analogue thereof.

Other suitable adjuvants may furthermore be selected from nucleic acidshaving the formula (VI): C_(l)X_(m)C_(n), wherein: C is cytosine, uracilor an analogue of cytosine or uracil; X is guanosine, uracil, adenosine,thymidine, cytosine or an analogue of the above-mentioned nucleotides; Iis an integer from 1 to 40, wherein when I=1 C is cytosine or ananalogue thereof, when I>1 at least 50% of the nucleotides are cytosineor an analogue thereof; m is an integer and is at least 3; wherein whenm=3 X is uracil or an analogue thereof, when m>3 at least 3 successiveuracils or analogues of uracil occur; n is an integer from 1 to 40,wherein when n=1 C is cytosine or an analogue thereof, when n>1 at least50% of the nucleotides are cytosine or an analogue thereof.

The inventive vaccine or composition can additionally contain one ormore auxiliary substances in order to further increase theimmunogenicity. A synergistic action of the artificial nucleic acid ofthe composition or vaccine as defined herein and of an auxiliarysubstance, which may be optionally be co-formulated (or separatelyformulated) with the inventive vaccine or composition as describedabove, is preferably achieved thereby. Depending on the various types ofauxiliary substances, various mechanisms can come into consideration inthis respect. For example, compounds that permit the maturation ofdendritic cells (DCs), for example lipopolysaccharides, TNF-alpha orCD40 ligand, form a first class of suitable auxiliary substances. Ingeneral, it is possible to use as auxiliary substance any agent thatinfluences the immune system in the manner of a “danger signal” (LPS,GP96, etc.) or cytokines, such as GM-CFS, which allow an immune responseproduced by the immune-stimulating adjuvant according to the inventionto be enhanced and/or influenced in a targeted manner. Particularlypreferred auxiliary substances are cytokines, such as monokines,lymphokines, interleukins or chemokines, that—additional to induction ofthe adaptive immune response by the encoded at least one antigen—promotethe innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta,INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors,such as hGH. Preferably, such immunogenicity increasing agents orcompounds are provided separately (not co-formulated with the inventivevaccine or composition) and administered individually.

The inventive vaccine or composition can also additionally contain anyfurther compound, which is known to be immune-stimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventive vaccineor composition in this context, may be CpG nucleic acids, in particularCpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-strandedCpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), asingle-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA,more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). TheCpG nucleic acid preferably contains at least one or more (mitogenic)cytosine/guanine dinucleotide sequence(s) (CpG motif(s)).

According to a first preferred alternative, at least one CpG motifcontained in these sequences, that is to say the C (cytosine) and the G(guanine) of the CpG motif, is unmethylated. All further cytosines orguanines optionally contained in these sequences can be eithermethylated or unmethylated. According to a further preferredalternative, however, the C (cytosine) and the B (guanine) of the CpGmotif can also be present in methylated form.

Preferably, the above compounds are formulated and administeredseparately from the above composition or vaccine (of the invention)containing the artificial nucleic acid according to the invention.

Polypeptide:

In a further aspect, the present invention concerns a polypeptideencoded by the inventive artificial nucleic acid as described herein, ora fragment of said polypeptide.

More preferably, the inventive polypeptide comprises or consists of,preferably in this order from N-terminus to C-terminus:

-   -   a) an amino acid sequence derived from a C-terminal fragment        from mature Norovirus capsid protein VP1, or a variant thereof,        wherein the C-terminal fragment preferably comprises or consists        of 3 to 20 amino acid residues,    -   b) an amino acid sequence derived from a signal sequence of        Norovirus capsid protein VP1, or a fragment or variant thereof,        or an amino acid sequence derived from a C-terminal fragment, or        a variant thereof, of Norovirus capsid protein VP1 as present in        Norovirus polyprotein before cleavage, preferably as described        herein.

More preferably, the inventive polypeptide is selected from the groupconsisting of Norovirus NS1/NS2, NS3, NS4, NS5, NS6, NS7, VP1, and VP2,or a fragment or variant of any of these proteins, and at least oneamino acid sequence selected from the group consisting of:

-   -   a) an amino acid sequence derived from a C-terminal fragment        from mature Norovirus capsid protein VP1, or a variant thereof,        wherein the C-terminal fragment consists of 3 to 20 amino acid        residues,    -   b) an amino acid sequence derived from a signal sequence of        Norovirus capsid protein VP1, or a fragment or variant thereof,        and    -   c) an amino acid sequence derived from an N-terminal fragment        from mature Norovirus non-structural protein NS1/NS2, NS3, NS4,        NS5, NS6, or NS7, or a variant thereof, wherein the N-terminal        fragment consists of 3 to 20 amino acid residues.

In a preferred embodiment, the inventive polypeptide does not comprisean amino acid sequence from Norovirus capsid protein VP1 or fromNorovirus non-structural protein 1 (NS1) distinct from the followingamino acid sequences:

-   -   a) an amino acid sequence derived from a C-terminal fragment        from mature Norovirus capsid protein VP1, or a variant thereof,        wherein the C-terminal fragment preferably comprises or consists        of 3 to 20 amino acid residues,    -   b) an amino acid sequence derived from a signal sequence of        Norovirus capsid protein VP1, or a fragment or variant thereof,        or an amino acid sequence derived from a C-terminal fragment, or        a variant thereof, of Norovirus capsid protein VP1 as present in        Norovirus polyprotein before cleavage, preferably as described        herein, and    -   c) an amino acid sequence derived from an N-terminal fragment        from mature Norovirus non-structural protein (NS1), or a variant        thereof, wherein the N-terminal fragment preferably comprises or        consists of 3 to 20 amino acid residues.

According to a preferred embodiment, the inventive polypeptides asdescribed herein comprises a molecular tag, wherein the molecular tag isselected from the group consisting of a FLAG tag, aglutathione-S-transferase (GST) tag, a His tag, a Myc tag, an E tag, aStrep tag, a green fluorescent protein (GFP) tag and an HA tag.

In a further aspect, the present invention provides a compositioncomprising at least one of the inventive polypeptides as describedherein. In a preferred embodiment, the inventive composition comprisesone type of polypeptide as described herein. Alternatively, theinventive composition may comprise at least two different inventivepolypeptides as described herein.

Preferably, the inventive composition comprises or consists of at leastone of the inventive polypeptides described herein and apharmaceutically acceptable carrier. In this context, thepharmaceutically acceptable carrier as well as optional furthercomponents of the composition are preferably as described herein withrespect to the inventive composition comprising at least one inventiveartificial nucleic acid.

In a further aspect, the invention concerns a vaccine comprising theinventive composition comprising at least one of the polypeptidesaccording to the invention. Therein, the at least one of the inventivepolypeptides preferably elicits an adaptive immune response uponadministration to a subject. More preferably, the vaccine according tothe invention comprising at least one of the inventive polypeptides orthe inventive composition comprising at least one of the polypeptidesaccording to the invention is preferably a vaccine as described herein.Reference is made to the respective description herein.

As used herein, the term ‘inventive composition’ may refer to theinventive composition comprising at least one artificial nucleic acidaccording to the invention as well as to the inventive compositioncomprising at least one of the polypeptides according to the invention.Likewise, the term ‘inventive vaccine’, as used in this context, mayrefer to an inventive vaccine, which is based on the inventiveartificial nucleic acid, i.e. which comprises at least one artificialnucleic acid according to the invention or which comprises the inventivecomposition comprising said artificial nucleic acid, as well as to aninventive vaccine, which is based on the inventive polypeptide(s), i.e.which comprises at least one polypeptide according to the invention orwhich comprises the inventive composition comprising said at least onepolypeptide according to the invention.

According to another embodiment, the present invention also provideskits, particularly kits of parts, comprising the artificial nucleic acidaccording to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide or the inventive vaccineas described herein, optionally a liquid vehicle for solubilising andoptionally technical instructions with information on the administrationand dosage of the artificial nucleic acid according as described herein,the inventive composition comprising at least one artificial nucleicacid according to the invention, the inventive polypeptides as describedherein, the inventive composition comprising at least one inventivepolypeptide or the inventive vaccine. The technical instructions maycontain information about administration and dosage. Such kits,preferably kits of parts, may be applied e.g. for any of theapplications or uses mentioned herein, preferably for the use of theartificial nucleic acid according as described herein, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, the inventive polypeptides as described herein, theinventive composition comprising at least one inventive polypeptide orthe inventive vaccine for the treatment or prophylaxis of a Norovirusinfection or diseases or disorders related thereto. The kits may also beapplied for the use of the artificial nucleic acid according asdescribed herein, the inventive composition comprising at least oneartificial nucleic acid according to the invention, the inventivepolypeptides as described herein, the inventive composition comprisingat least one inventive polypeptide or the inventive vaccine for thetreatment or prophylaxis of Norovirus infection or diseases or disordersrelated thereto, wherein the artificial nucleic acid according asdescribed herein, the inventive composition comprising at least oneartificial nucleic acid according to the invention, the inventivepolypeptides as described herein, the inventive composition comprisingat least one inventive polypeptide or the inventive vaccine may induceor enhance an immune response in a mammal as defined above. Preferably,the artificial nucleic acid according as described herein, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, or the inventive vaccine is provided in a separate partof the kit, wherein the artificial nucleic acid according as describedherein, the inventive composition comprising at least one artificialnucleic acid according to the invention, or the inventive vaccine arepreferably lyophilised. More preferably, the kit further contains as apart a vehicle for solubilising the artificial nucleic acid according asdescribed herein, the inventive composition comprising at least oneartificial nucleic acid according to the invention, or the inventivevaccine, the vehicle preferably being Ringer-lactate solution. Any ofthe above kits may be used in a treatment or prophylaxis as definedabove. More preferably, any of the above kits may be used as a vaccine,preferably a vaccine against Norovirus infection or a related disease ordisorder.

The present invention furthermore provides several applications and usesof the artificial nucleic acid according to the invention, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, the inventive polypeptides as described herein, theinventive composition comprising at least one inventive polypeptide orthe inventive vaccine or of kits comprising same. In particular, theinventive (pharmaceutical) composition(s) or the inventive vaccine maybe used for human and also for veterinary medical purposes, preferablyfor human medical purposes, as a pharmaceutical composition in generalor as a vaccine.

In a further aspect, the invention provides the artificial nucleic acidaccording to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide, the inventive vaccine orthe inventive kit or kit of parts for use in a method of prophylactic(pre-exposure prophylaxis or post-exposure prophylaxis) and/ortherapeutic treatment of Norovirus infections. Consequently, in afurther aspect, the present invention is directed to the first medicaluse of the artificial nucleic acid according to the invention, theinventive composition comprising at least one artificial nucleic acidaccording to the invention, the inventive polypeptides as describedherein, the inventive composition comprising at least one inventivepolypeptide, the inventive vaccine or the inventive kit or kit of partsas defined herein as a medicament. Particularly, the invention providesthe use of an artificial nucleic acid comprising at least one codingregion encoding at least one polypeptide comprising at least oneNorovirus protein as defined herein, or a fragment or variant thereof asdescribed herein for the preparation of a medicament.

According to another aspect, the present invention is directed to thesecond medical use of the artificial nucleic acid according to theinvention, the inventive composition comprising at least one artificialnucleic acid according to the invention, the inventive polypeptides asdescribed herein, the inventive composition comprising at least oneinventive polypeptide, the inventive vaccine or the inventive kit or kitof parts for the treatment of an infection with Norovirus or a diseaseor disorder related to an infection with Norovirus as defined herein.Particularly, the artificial nucleic acid comprising at least one codingregion encoding at least one polypeptide comprising at least oneNorovirus protein as defined herein, or a fragment or variant thereof asdescribed herein to be used in a method as said above is an artificialnucleic acid formulated together with a pharmaceutically acceptablevehicle and an optionally additional adjuvant and an optionallyadditional further component as defined herein.

The invention provides the artificial nucleic acid according to theinvention, the inventive composition comprising at least one artificialnucleic acid according to the invention, the inventive polypeptides asdescribed herein, the inventive composition comprising at least oneinventive polypeptide, the inventive vaccine or the inventive kit or kitof parts for medical use, in particular for the treatment of aninfection with Norovirus or a disease or disorder related to aninfection with Norovirus, wherein preferably an infection with Norovirusmay involve any Norovirus strain. More preferably, the Norovirusinfection is caused by a Norovirus strain, which is selected from thegroup consisting of GII.4 CIN-1 Norovirus or a GII.4 Sydney Norovirus,GI.1 and GII.4 Sydney 2012 Norovirus. Further preferably, the Norovirusinfection is caused by a Norovirus strain GII.4 Sydney or GII.4 Sydney2012.

As used herein, “a disorder related to a Norovirus infection” or “adisease related to a Norovirus infection” may preferably comprise acomplication of Norovirus infection, such as abdominal pain, diarrhea,DIC (disseminated intravascular coagulation), fever, fever/chills,gastrointestinal symptoms, headache, nausea, neck stiffness,obtundation, photophobia, and/or vomiting. In a preferred embodiment,the inventive composition or vaccine is thus used for treatment orprophylaxis, preferably prophylaxis, of complications associated with aNorovirus infection.

The inventive composition or the inventive vaccine, in particular theinventive composition comprising at least one artificial nucleic acidaccording to the invention, the inventive polypeptides as describedherein or the inventive composition comprising at least one inventivepolypeptide, can be administered, for example, systemically or locally.Routes for systemic administration in general include, for example,transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Inventive vaccines are therefore preferablyformulated in liquid (or sometimes in solid) form. Preferably, theinventive vaccine may be administered by conventional needle injectionor needle-free jet injection. In a preferred embodiment the inventivevaccine or composition may be administered by jet injection as definedherein, preferably intramuscularly or intradermally, more preferablyintradermally.

In a preferred embodiment, a single dose of the inventive artificialnucleic acid, composition or vaccine comprises a specific amount of theartificial nucleic acid according to the invention. Preferably, theinventive artificial nucleic acid is provided in an amount of at least10 μg per dose, 40 μg per dose, preferably in an amount of from 40 to700 μg per dose, more preferably in an amount of from BD to 400 μg perdose. More specifically, in the case of intradermal injection, which ispreferably carried out by using a conventional needle, the amount of theinventive artificial nucleic acid comprised in a single dose istypically at least 200 μg, preferably from 200 μg to 1.000 μg, morepreferably from 300 μg to 850 μg, even more preferably from 300 μg to700 μg. In the case of intradermal injection, which is preferablycarried out via jet injection (e.g. using a Tropis device), the amountof the inventive artificial nucleic acid comprised in a single dose istypically at least 80 μg, preferably from 80 μg to 700 μg, morepreferably from 80 μg to 400 μg. Moreover, in the case of intramuscularinjection, which is preferably carried out by using a conventionalneedle or via jet injection, the amount of the inventive artificialnucleic acid comprised in a single dose is typically at least 80 μg,preferably from 80 μg to 1000 μg, more preferably from BO μg to 850 μg,even more preferably from 80 μg to 700 μg.

Depending on the used formulation, the used route of application, anddepending on the subject (human, animal), the dose of the inventiveartificial nucleic acid may range from about 1 μg to about 1000 μg,preferably from about 10 μg to about 500 μg.

The immunization protocol for the treatment or prophylaxis of aNorovirus infection, i.e the immunization of a subject againstNorovirus, typically comprises a series of single doses or dosages ofthe inventive composition or the inventive vaccine. A single dosage, asused herein, refers to the initial/first dose, a second dose or anyfurther doses, respectively, which are preferably administered in orderto “boost” the immune reaction.

According to a preferred embodiment, the artificial nucleic acidaccording to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide, the inventive vaccine orthe inventive kit or kit of parts is provided for use in treatment orprophylaxis, preferably treatment or prophylaxis of a Norovirusinfection or a related disorder or disease, wherein the treatment orprophylaxis comprises the administration of a further activepharmaceutical ingredient. More preferably, in the case of the inventivevaccine or composition, which is based on the inventive artificialnucleic acid, a polypeptide may be co-administered as a further activepharmaceutical ingredient. For example, at least one Norovirus proteinas described herein, or a fragment or variant thereof, may beco-administered in order to induce or enhance an immune response.Likewise, in the case of the inventive vaccine or composition, which isbased on the inventive polypeptide as described herein, an artificialnucleic acid as described herein may be co-administered as a furtheractive pharmaceutical ingredient. For example, an artificial nucleicacid as described herein encoding at least one polypeptide as describedherein may be co-administered in order to induce or enhance an immuneresponse.

A further component of the inventive vaccine or composition may be animmunotherapeutic agent that can be selected from immunoglobulins,preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serumor sera, etc, most preferably immunoglobulins directed against aNorovirus. Preferably, such a further immunotherapeutic agent may beprovided as a peptide/protein or may be encoded by a nucleic acid,preferably by a DNA or an RNA, more preferably an mRNA. Such animmunotherapeutic agent allows providing passive vaccination additionalto active vaccination triggered by the inventive artificial nucleic acidor by the inventive polypeptide.

In a further aspect the invention provides a method of treating orpreventing a disorder, wherein the disorder is preferably an infectionwith Norovirus or a disorder related to an infection with Norovirus,wherein the method comprises administering to a subject in need thereofthe artificial nucleic acid according to the invention, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, the inventive polypeptides as described herein, theinventive composition comprising at least one inventive polypeptide, theinventive vaccine or the inventive kit or kit of parts.

In particular, such a method may preferably comprise the steps of:

-   -   a) providing the artificial nucleic acid according to the        invention, the inventive composition comprising at least one        artificial nucleic acid according to the invention, the        inventive polypeptides as described herein, the inventive        composition comprising at least one inventive polypeptide, the        inventive vaccine or the inventive kit or kit of parts;    -   b) applying or administering the artificial nucleic acid        according to the invention, the inventive composition comprising        at least one artificial nucleic acid according to the invention,        the inventive polypeptides as described herein, the inventive        composition comprising at least one inventive polypeptide, the        inventive vaccine or the inventive kit or kit of parts to a        tissue or an organism;    -   c) optionally administering immune globuline against Norovirus.

According to a further aspect, the present invention also provides amethod for expression of at least one polypeptide comprising at leastone Norovirus, or a fragment or variant thereof, wherein the methodpreferably comprises the following steps:

-   -   a) providing the inventive artificial nucleic acid comprising at        least one coding region encoding at least one polypeptide        comprising at least one Norovirus, or a fragment or variant        thereof, preferably as defined herein, or a composition        comprising said artificial nucleic acid: and    -   b) applying or administering the inventive artificial nucleic        acid or the inventive composition comprising said artificial        nucleic acid to an expression system, e.g. to a cell-free        expression system, a cell (e.g. an expression host cell or a        somatic cell), a tissue or an organism.

The method may be applied for laboratory, for research, for diagnostic,for commercial production of peptides or proteins and/or for therapeuticpurposes. In this context, typically after preparing the inventiveartificial nucleic acid as defined herein or of the inventivecomposition or vaccine as defined herein, it is typically applied oradministered to a cell-free expression system, a cell (e.g. anexpression host cell or a somatic cell), a tissue or an organism, e.g.in naked or complexed form or as a (pharmaceutical) composition orvaccine as described herein, preferably via transfection or by using anyof the administration modes as described herein. The method may becarried out in vitro, in viva or ex viva. The method may furthermore becarried out in the context of the treatment of a specific disease,particularly in the treatment of infectious diseases, preferablyNorovirus infection or a related disorder as defined herein.

In this context, in vitro is defined herein as transfection ortransduction of the inventive artificial nucleic acid as defined hereinor of the inventive composition or vaccine as defined herein into cellsin culture outside of an organism; in viva is defined herein astransfection or transduction of the inventive artificial nucleic acid orof the inventive composition or vaccine into cells by application of theinventive mRNA or of the inventive composition to the whole organism orindividual and ex vivo is defined herein as transfection or transductionof the inventive artificial nucleic acid or of the inventive compositionor vaccine into cells outside of an organism or individual andsubsequent application of the transfected cells to the organism orindividual.

Likewise, according to another aspect, the present invention alsoprovides the use of the inventive artificial nucleic acid as definedherein or of the inventive composition or vaccine as defined herein,preferably for diagnostic or therapeutic purposes, for expression of anencoded antigenic peptide or protein, e.g. by applying or administeringthe inventive artificial nucleic acid as defined herein or of theinventive composition or vaccine as defined herein, e.g. to a cell-freeexpression system, a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism. The use may be applied for a(diagnostic) laboratory, for research, for diagnostics, for commercialproduction of peptides or proteins and/or for therapeutic purposes. Inthis context, typically after preparing the inventive artificial nucleicacid as defined herein or of the inventive composition or vaccine asdefined herein, it is typically applied or administered to a cell-freeexpression system, a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism, preferably in naked form or complexedform, or as a (pharmaceutical) composition or vaccine as describedherein, preferably via transfection or by using any of theadministration modes as described herein. The use may be carried out invitro, in vivo or ex vivo. The use may furthermore be carried out in thecontext of the treatment of a specific disease, particularly in thetreatment of Norovirus infection or a related disorder.

In a particularly preferred embodiment, the invention provides theartificial nucleic acid, the inventive composition or the inventivevaccine for use as defined herein, preferably for use as a medicament,for use in treatment or prophylaxis, preferably treatment or prophylaxisof a Norovirus infection or a related disorder, or for use as a vaccine.The vaccine or composition according to the invention can be usedaccording to the invention for human and also for veterinary medicalpurposes (mammals, vertebrates), as a pharmaceutical composition or as avaccine.

Adjuvants:

According to another embodiment, the (pharmaceutical) composition orvaccine according to the invention may comprise an adjuvant, which ispreferably added in order to enhance the immunostimulatory properties ofthe composition. In this context, an adjuvant may be understood as anycompound, which is suitable to support administration and delivery ofthe composition according to the invention. Furthermore, such anadjuvant may, without being bound thereto, initiate or increase animmune response of the innate immune system, i.e. a non-specific immuneresponse. In other words, when administered, the composition accordingto the invention typically initiates an adaptive immune response due toan antigen as defined herein or a fragment or variant thereof, which isencoded by the at least one coding sequence of the inventive mRNAcontained in the composition of the present invention. Additionally, thecomposition according to the invention may generate an (supportive)innate immune response due to addition of an adjuvant as defined hereinto the composition according to the invention.

Particularly preferred, an adjuvant may be selected from adjuvants,which support induction of a Th1-immune response or maturation of naïveT-cells, such as GM-CSF, IL-12, IFN0, any immunostimulatory nucleic acidas defined above, preferably an immunostimulatory RNA, CpG DNA, etc.

In a further preferred embodiment it is also possible that the inventivecomposition contains besides the antigen-providing mRNA furthercomponents which are selected from the group comprising: furtherantigens (e.g. in the form of a peptide or protein) or furtherantigen-encoding nucleic acids; a further immunotherapeutic agent; oneor more auxiliary substances; or any further compound, which is known tobe immunostimulating due to its binding affinity (as ligands) to humanToll-like receptors; and/or an adjuvant nucleic acid, preferably animmunostimulatory RNA (isRNA).

The composition of the present invention can additionally contain one ormore auxiliary substances in order to increase its immunogenicity orimmunostimulatory capacity, if desired. A synergistic action of the mRNAas defined herein and of an auxiliary substance, which may be optionallycontained in the inventive composition, is preferably achieved thereby.Depending on the various types of auxiliary substances, variousmechanisms can come into consideration in this respect. For example,compounds that permit the maturation of dendritic cells (DCs), forexample lipopolysaccharides, TNF-alpha or CD40 ligand, form a firstclass of suitable auxiliary substances. In general, it is possible touse as auxiliary substance any agent that influences the immune systemin the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, suchas GM-CFS, which allow an immune response to be enhanced and/orinfluenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that further promote the innate immune response, such asIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta orTNF-alpha, growth factors, such as hGH.

Suitable adjuvants may also be selected from cationic or polycationiccompounds wherein the adjuvant is preferably prepared upon complexingthe mRNA of the composition according to the invention with the cationicor polycationic compound. Associating or complexing the mRNA of thecomposition with cationic or polycationic compounds as defined hereinpreferably provides adjuvant properties and confers a stabilizing effectto the mRNA of the composition. In particular, such preferred cationicor polycationic compounds are selected from cationic or polycationicpeptides or proteins, including protamine, nucleoline, spermin orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpTG20, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine,spermine, spermidine, or histones. Further preferred cationic orpolycationic compounds may include cationic polysaccharides, for examplechitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI),cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected of a cationic polymer as mentioned above) and of one ormore hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); etc.

Additionally, preferred cationic or polycationic proteins or peptides,which can be used as an adjuvant by complexing the mRNA of thecomposition according to the invention, may be selected from followingproteins or peptides having the following total formula (III): (Arg)l;(Lys)m; (His)n; (Orn)o; (Xaa)x, wherein l+m+n+o+x=8-15, and l, m, n or oindependently of each other may be any number selected from 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overallcontent of Arg, Lys, His and Orn represents at least 50% of all aminoacids of the oligopeptide; and Xaa may be any amino acid selected fromnative (=naturally occurring) or non-native amino acids except of Arg,Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4,provided, that the overall content of Xaa does not exceed 50% of allamino acids of the oligopeptide. Particularly preferred oligoargininesin this context are e.g. Arg7, Arg8, Arg9, Arg7, H3R9, R9H3, H3R9H3,YSSR9SSY, (RKH)4, Y(RKH)2R, etc.

The ratio of the mRNA to the cationic or polycationic compound in theadjuvant component may be calculated on the basis of thenitrogen/phosphate ratio (N/P-ratio) of the entire mRNA complex, i.e.the ratio of positively charged (nitrogen) atoms of the cationic orpolycationic compound to the negatively charged phosphate atoms of thenucleic acids. For example, 1 μg of RNA typically contains about 3 nmolphosphate residues, provided the RNA exhibits a statistical distributionof bases. Additionally, 1 μg of peptide typically contains about x nmolnitrogen residues, dependent on the molecular weight and the number ofbasic amino acids. When exemplarily calculated for (Arg)9 (molecularweight 1424 g/mol, 9 nitrogen atoms), 1 μg (Arg)9 contains about 700pmol (Arg)9 and thus 700×9=6300 pmol basic amino acids=6.3 nmol nitrogenatoms. For a mass ratio of about 1:1 RNA/(Arg)9 an N/P ratio of about 2can be calculated. When exemplarily calculated for protamine (molecularweight about 4250 g/mol, 21 nitrogen atoms, when protamine from salmonis used) with a mass ratio of about 2:1 with 2 Ξg RNA, 6 nmol phosphateare to be calculated for the RNA: 1 μg protamine contains about 235 pmolprotamine molecules and thus 235×21=4935 pmol basic nitrogen atoms=4.9nmol nitrogen atoms. For a mass ratio of about 2:1 RNA/protamine an N/Pratio of about 0.81 can be calculated. For a mass ratio of about 8:1RNA/protamine an N/P ratio of about 0.2 can be calculated. In thecontext of the present invention, an N/P-ratio is preferably in therange of about 0.1-10, preferably in a range of about 0.3-4 and mostpreferably in a range of about 0.5-2 or 0.7-2 regarding the ratio ofRNA:peptide in the complex, and most preferably in the range of about0.7-1.5.

In a preferred embodiment, the composition of the present invention isobtained in two separate steps in order to obtain both, an efficientimmunostimulatory effect and efficient translation of the mRNA accordingto the invention. Therein, a so called “adjuvant component” is preparedby complexing in a first step an mRNA as defined herein of the adjuvantcomponent with a cationic or polycationic compound in a specific ratioto form a stable complex. In this context, it is important, that no freecationic or polycationic compound or only a negligibly small amountremains in the adjuvant component after complexing the mRNA.Accordingly, the ratio of the mRNA and the cationic or polycationiccompound in the adjuvant component is typically selected in a range thatthe mRNA is entirely complexed and no free cationic or polycationiccompound or only a negligible small amount remains in the composition.Preferably the ratio of the adjuvant component, i.e. the ratio of themRNA to the cationic or polycationic compound is selected from a rangeof about 9:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1(w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) toabout 1:1 (w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and mostpreferably a ratio of about 3:1 (w/w) to about 2:1 (w/w).

According to a preferred embodiment, the mRNA of the inventioncomprising at least one mRNA sequence comprising at least one codingregion as defined herein is added in a second step to the complexed mRNAof the adjuvant component in order to form the (immunostimulatory)composition of the invention. Therein, the mRNA of the compositionaccording to the invention is added as free mRNA, which is not complexedby other compounds. Prior to addition, the free mRNA is not complexedand will preferably not undergo any detectable or significantcomplexation reaction upon the addition of the adjuvant component. Thisis due to the strong binding of the cationic or polycationic compound tothe above described mRNA according to the invention comprised in theadjuvant component. In other words, when the mRNA comprising at leastone coding region as defined herein is added to the “adjuvantcomponent”, preferably no free or substantially no free cationic orpolycationic compound is present, which could form a complex with thefree mRNA. Accordingly, an efficient translation of the mRNA of thecomposition is possible in vivo. Therein, the free mRNA, may occur as amono-, di-, or multicistronic mRNA, i.e. an mRNA which carries thecoding sequences of one or more proteins. Such coding sequences in di-,or even multicistronic mRNA may be separated by at least one IRESsequence, e.g. as defined herein.

In a particularly preferred embodiment, the free mRNA as defined herein,which is comprised in the composition of the present invention, may beidentical or different to the RNA as defined herein, which is comprisedin the adjuvant component of the composition, depending on the specificrequirements of therapy. Even more preferably, the free RNA, which iscomprised in the composition according to the invention, is identical tothe RNA of the adjuvant component of the inventive composition.

In a particularly preferred embodiment, the composition according to theinvention comprises the mRNA of the invention, which encodes at leastone antigenic peptide or protein as defined herein and wherein said mRNAis present in the composition partially as free mRNA and partially ascomplexed mRNA. Preferably, the mRNA as defined herein is complexed asdescribed above and the same mRNA is then added as free mRNA, whereinpreferably the compound, which is used for complexing the mRNA is notpresent in free form in the composition at the moment of addition of thefree mRNA component.

The ratio of the first component (i.e. the adjuvant component comprisingor consisting of the mRNA as defined herein complexed with a cationic orpolycationic compound) and the second component (i.e. the free mRNA asdefined herein) may be selected in the inventive composition accordingto the specific requirements of a particular therapy. Typically, theratio of the mRNA in the adjuvant component and the at least one freemRNA (mRNA in the adjuvant component:free mRNA) of the compositionaccording to the invention is selected such that a significantstimulation of the innate immune system is elicited due to the adjuvantcomponent. In parallel, the ratio is selected such that a significantamount of the free mRNA can be provided in vivo leading to an efficienttranslation and concentration of the expressed protein in vivo, e.g. theat least one antigenic peptide or protein as defined herein. Preferablythe ratio of the mRNA in the adjuvant component:free mRNA in theinventive composition is selected from a range of about 5:1 (w/w) toabout 1:10 (w/w), more preferably from a range of about 4:1 (w/w) toabout 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) toabout 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of mRNA inthe adjuvant component:free mRNA in the inventive composition isselected from a ratio of about 1:1 (w/w).

Additionally or alternatively, the ratio of the first component (i.e.the adjuvant component comprising or consisting of the mRNA complexedwith a cationic or polycationic compound) and the second component (i.e.the free mRNA) may be calculated on the basis of the nitrogen/phosphateratio (N/P-ratio) of the entire mRNA complex. In the context of thepresent invention, an N/P-ratio is preferably in the range of about0.1-10, preferably in a range of about 0.3-4 and most preferably in arange of about 0.5-2 or 0.7-2 regarding the ratio of mRNA:peptide in thecomplex, and most preferably in the range of about 0.7-1.5.

Additionally or alternatively, the ratio of the first component (i.e.the adjuvant component comprising or consisting of the mRNA complexedwith a cationic or polycationic compound) and the second component (i.e.the free mRNA) may also be selected in the composition according to theinvention on the basis of the molar ratio of both mRNAs to each other,i.e. the mRNA of the adjuvant component, being complexed with a cationicor polycationic compound and the free mRNA of the second component.Typically, the molar ratio of the mRNA of the adjuvant component to thefree mRNA of the second component may be selected such, that the molarratio suffices the above (w/w) and/or N/P-definitions. More preferably,the molar ratio of the mRNA of the adjuvant component to the free mRNAof the second component may be selected e.g. from a molar ratio of about0.001:1, 0.01:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 116:1, 0.7:1, 0.8:1,0.9:1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2,1:0.1, 1:0.01, 1:0.001, etc. or from any range formed by any two of theabove values, e.g. a range selected from about 0.001:1 to 1:0.001,including a range of about 0.01:1 to 1:0.001, 0.1:1 to 1:0.001, 0.2:1 to1:0.001, 0.3:1 to 1:0.001, 0.4:1 to 1:0.001, 0.5:1 to 1:0.001, 0.6:1 to1:0.001, 0.7:1 to 1:0.001, 0.8:1 to 1:0.001, 0.9:1 to 1:0.001, 1:1 to1:0.001, 1:0.9 to 1:0.001, 1:0.8 to 1:0.001, 1:0.7 to 1:0.001, 1:0.6 to1:0.001, 1:0.5 to 1:0.001, 1:0.4 to 1:0.001, 1:0.3 to 1:0.001, 1:0.2 to1:0.001, 1:0.1 to 1:0.001, 1:0.01 to 1:0.001, or a range of about 0.01:1to 1:0.01, 0.1:1 to 1:0.01, 0.2:1 to 1:0.01, 0.3:1 to 1:0.01, 0.4:1 to1:0.01, 0.5:1 to 1:0.01, 0.6:1 to 1:0.01, 0.7:1 to 1:0.01, 0.8:1 to1:0.01, 0.9:1 to 1:0.01, 1:1 to 1:0.01, 1:0.9 to 1:0.01, 1:0.8 to1:0.01, 1:0.7 to 1:0.01, 1:0.6 to 1:0.01, 1:0.5 to 1:0.01, 1:0.4 to1:0.01, 1:0.3 to 1:0.01, 1:0.2 to 1:0.01, 1:0.1 to 1:0.01, 1:0.01 to1:0.01, or including a range of about 0.001:1 to 1:0.01, 0.001:1 to1:0.1, 0.001:1 to 1:0.2, 0.001:1 to 1:0.3, 0.001:1 to 1:0.4, 0.001:1 to1:0.5, 0.001:1 to 1:0.6, 0.001:1 to 1:0.7, 0.001:1 to 1:0.8, 0.001:1 to1:0.9, 0.001:1 to 1:1, 0.001 to 0.9:1, 0.001 to 0.8:1, 0.001 to 0.7:1,0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to 0.4:1, 0.001 to 0.3:1, 0.001 to0.2:1, 0.001 to 0.1:1, or a range of about 0.01:1 to 1:0.01, 0.01:1 to1:0.1, 0.01:1 to 1:0.2, 0.01:1 to 1:0.3, 0.01:1 to 1:0.4, 0.01:1 to1:0.5, 0.01:1 to 1:0.6, 0.01:1 to 1:0.7, 0.01:1 to 1:0.8, 0.01:1 to1:0.9, 0.01:1 to 1:1, 0.001 to 0.9:1, 0.001 to 0.8:1, 0.001 to 0.7:1,0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to 0.4:1, 0.001 to 0.3:1, 0.001 to0.2:1, 0.001 to 0.1:1, etc.

Even more preferably, the molar ratio of the mRNA of the adjuvantcomponent to the free mRNA of the second component may be selected e.g.from a range of about 0.01:1 to 1:0.01. Most preferably, the molar ratioof the mRNA of the adjuvant component to the free mRNA of the secondcomponent may be selected e.g. from a molar ratio of about 1:1. Any ofthe above definitions with regard to (w/w) and/or N/P ratio may alsoapply.

Suitable adjuvants may furthermore be selected from nucleic acids havingthe formula (Va): GIXmGn, wherein: G is guanosine, uracil or an analogueof guanosine or uracil; X is guanosine, uracil, adenosine, thymidine,cytosine or an analogue of the above-mentioned nucleotides; I is aninteger from 1 to 40, wherein when I=1 G is guanosine or an analoguethereof, when I>1 at least 50% of the nucleotides are guanosine or ananalogue thereof; m is an integer and is at least 3; wherein when m=3 Xis uracil or an analogue thereof, when m>3 at least 3 successive uracilsor analogues of uracil occur; n is an integer from 1 to 40, wherein whenn=1 G is guanosine or an analogue thereof, when n>1 at least 50% of thenucleotides are guanosine or an analogue thereof, or formula (Vb):(NuGIXmGnNv)a, wherein: G is guanosine (guanine), uridine (uracil) or ananalogue of guanosine (guanine) or uridine (uracil), preferablyguanosine (guanine) or an analogue thereof; X is guanosine (guanine),uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine(cytosine), or an analogue of these nucleotides (nucleosides),preferably uridine (uracil) or an analogue thereof; N is a nucleic acidsequence having a length of about 4 to 50, preferably of about 4 to 40,more preferably of about 4 to 30 or 4 to 20 nucleic acids, each Nindependently being selected from guanosine (guanine), uridine (uracil),adenosine (adenine), thymidine (thymine), cytidine (cytosine) or ananalogue of these nucleotides (nucleosides); a is an integer from 1 to20, preferably from 1 to 15, most preferably from 1 to 10; I is aninteger from 1 to 40, wherein when I=1 G is guanosine (guanine) or ananalogue thereof, when I>1, at least 50% of these nucleotides(nucleosides) are guanosine (guanine) or an analogue thereof; m is aninteger and is at least 3; wherein when m=3. X is uridine (uracil) or ananalogue thereof, and when m>3, at least 3 successive uridines (uracils)or analogues of uridine (uracil) occur; n is an integer from 1 to 40,wherein when n=1, G is guanosine (guanine) or an analogue thereof, whenn>1, at least 50% of these nucleotides (nucleosides) are guanosine(guanine) or an analogue thereof; u,v may be independently from eachother an integer from 0 to 50, preferably wherein when u=0, v≥1, or whenv=0, u≥1; wherein the nucleic acid molecule of formula (Vb) has a lengthof at least 50 nucleotides, preferably of at least 100 nucleotides, morepreferably of at least 150 nucleotides, even more preferably of at least200 nucleotides and most preferably of at least 250 nucleotides.

Other suitable adjuvants may furthermore be selected from nucleic acidshaving the formula (VI): CIXmCn, wherein: C is cytosine, uracil or ananalogue of cytosine or uracil; X is guanosine, uracil, adenosine,thymidine, cytosine or an analogue of the above-mentioned nucleotides; Iis an integer from 1 to 40, wherein when I=1 C is cytosine or ananalogue thereof, when I>1 at least 50% of the nucleotides are cytosineor an analogue thereof; m is an integer and is at least 3; wherein whenm=3 X is uracil or an analogue thereof, when m>3 at least 3 successiveuracils or analogues of uracil occur; n is an integer from 1 to 40,wherein when n=1 C is cytosine or an analogue thereof, when n>1 at least50% of the nucleotides are cytosine or an analogue thereof.

In this context the disclosure of WO 2003/014979 and WO 2009/095229 isalso incorporated herein by reference.

In a further aspect, the present invention provides a vaccine, which isbased on the mRNA sequence according to the invention comprising atleast one coding region as defined herein. The vaccine according to theinvention is preferably a (pharmaceutical) composition as definedherein.

Accordingly, the vaccine according to the invention is based on the samecomponents as the (pharmaceutical) composition described herein.Insofar, it may be referred to the description of the (pharmaceutical)composition as provided herein. Preferably, the vaccine according to theinvention comprises at least one mRNA comprising at least one mRNAsequence as defined herein and a pharmaceutically acceptable carrier. Inembodiments, where the vaccine comprises more than one mRNA sequence(such as a plurality of RNA sequences according to the invention,wherein each preferably encodes a distinct antigenic peptide orprotein), the vaccine may be provided in physically separate form andmay be administered by separate administration steps. The vaccineaccording to the invention may correspond to the (pharmaceutical)composition as described herein, especially where the mRNA sequences areprovided by one single composition. However, the inventive vaccine mayalso be provided physically separated. For instance, in embodiments,wherein the vaccine comprises more than one mRNA sequences/species,these RNA species may be provided such that, for example, two, three,four, five or six separate compositions, which may contain at least onemRNA species/sequence each (e.g. three distinct mRNA species/sequences),each encoding distinct antigenic peptides or proteins, are provided,which may or may not be combined. Also, the inventive vaccine may be acombination of at least two distinct compositions, each compositioncomprising at least one mRNA encoding at least one of the antigenicpeptides or proteins defined herein. Alternatively, the vaccine may beprovided as a combination of at least one mRNA, preferably at least two,three, four, five, six or more mRNAs, each encoding one of the antigenicpeptides or proteins defined herein. The vaccine may be combined toprovide one single composition prior to its use or it may be used suchthat more than one administration is required to administer the distinctmRNA sequences/species encoding any of the antigenic peptides orproteins as defined herein. If the vaccine contains at least one mRNAsequence, typically at least two mRNA sequences, encoding the antigencombinations defined herein, it may e.g. be administered by one singleadministration (combining all mRNA species/sequences), by at least twoseparate administrations. Accordingly; any combination of mono-, bi- ormulticistronic mRNAs encoding the at least one antigenic peptide orprotein or any combination of antigens as defined herein (and optionallyfurther antigens), provided as separate entities (containing one mRNAspecies) or as combined entity (containing more than one mRNA species),is understood as a vaccine according to the present invention. Accordingto a particularly preferred embodiment of the inventive vaccine, the atleast one antigen, preferably a combination as defined herein of atleast two, three, four, five, six or more antigens encoded by theinventive composition as a whole, is provided as an individual(monocistronic) mRNA, which is administered separately.

As with the (pharmaceutical) composition according to the presentinvention, the entities of the vaccine may be provided in liquid and orin dry (e.g. lyophilized) form. They may contain further components, inparticular further components allowing for its pharmaceutical use. Thevaccine or the (pharmaceutical) composition may, e.g., additionallycontain a pharmaceutically acceptable carrier and/or further auxiliarysubstances and additives and/or adjuvants.

The vaccine or (pharmaceutical) composition typically comprises a safeand effective amount of the mRNA according to the invention as definedherein, encoding an antigenic peptide or protein as defined herein or afragment or variant thereof or a combination of antigens, preferably asdefined herein. As used herein, “safe and effective amount” means anamount of the mRNA that is sufficient to significantly induce a positivemodification of cancer or a disease or disorder related to cancer. Atthe same time, however, a “safe and effective amount” is small enough toavoid serious side-effects, that is to say to permit a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment. Inrelation to the vaccine or (pharmaceutical) composition of the presentinvention, the expression “safe and effective amount” preferably meansan amount of the mRNA (and thus of the encoded antigen) that is suitablefor stimulating the adaptive immune system in such a manner that noexcessive or damaging immune reactions are achieved but, preferably,also no such immune reactions below a measurable level. Such a “safe andeffective amount” of the mRNA of the (pharmaceutical) composition orvaccine as defined herein may furthermore be selected in dependence ofthe type of mRNA, e.g. monocistronic, bi- or even multicistronic mRNA,since a bi- or even multicistronic mRNA may lead to a significantlyhigher expression of the encoded antigen(s) than the use of an equalamount of a monocistronic mRNA. A “safe and effective amount” of themRNA of the (pharmaceutical) composition or vaccine as defined abovewill furthermore vary in connection with the particular condition to betreated and also with the age and physical condition of the patient tobe treated, the severity of the condition, the duration of thetreatment, the nature of the accompanying therapy, of the particularpharmaceutically acceptable carrier used, and similar factors, withinthe knowledge and experience of the accompanying doctor. The vaccine orcomposition according to the invention can be used according to theinvention for human and also for veterinary medical purposes, as apharmaceutical composition or as a vaccine.

In a preferred embodiment, the mRNA of the (pharmaceutical) composition,vaccine or kit of parts according to the invention is provided inlyophilized form. Preferably, the lyophilized mRNA is reconstituted in asuitable buffer, advantageously based on an aqueous carrier, prior toadministration, e.g. Ringer-Lactate solution, which is preferred, Ringersolution, a phosphate buffer solution. In a preferred embodiment, the(pharmaceutical) composition, the vaccine or the kit of parts accordingto the invention contains at least one, two, three, four, five, six ormore mRNAs, preferably mRNAs which are provided separately inlyophilized form (optionally together with at least one furtheradditive) and which are preferably reconstituted separately in asuitable buffer (such as Ringer-Lactate solution) prior to their use soas to allow individual administration of each of the (monocistronic)mRNAs.

The vaccine or (pharmaceutical) composition according to the inventionmay typically contain a pharmaceutically acceptable carrier. Theexpression “pharmaceutically acceptable carrier” as used hereinpreferably includes the liquid or non-liquid basis of the inventivevaccine. If the inventive vaccine is provided in liquid form, thecarrier will be water, typically pyrogen-free water;

isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrateetc. buffered solutions. Particularly for injection of the inventivevaccine, water or preferably a buffer, more preferably an aqueousbuffer, may be used, containing a sodium salt, preferably at least 50 mMof a sodium salt, a calcium salt, preferably at least 0.01 mM of acalcium salt, and optionally a potassium salt, preferably at least 3 mMof a potassium salt. According to a preferred embodiment, the sodium,calcium and, optionally, potassium salts may occur in the form of theirhalogenides, e.g. chlorides, iodides, or bromides, in the form of theirhydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Withoutbeing limited thereto, examples of sodium salts include e.g. NaCl, NaI,NaBr, Na2CO3, NaHCO3, Na2SO4, examples of the optional potassium saltsinclude e.g. KCl, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calciumsalts include e.g. CaCl2, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2.Furthermore, organic anions of the aforementioned cations may becontained in the buffer. According to a more preferred embodiment, thebuffer suitable for injection purposes as defined above, may containsalts selected from sodium chloride (NaCl), calcium chloride (CaCl2) andoptionally potassium chloride (KCl), wherein further anions may bepresent additional to the chlorides. CaCl2 can also be replaced byanother salt like KCl. Typically, the salts in the injection buffer arepresent in a concentration of at least 50 mM sodium chloride (NaCl), atleast 3 mM potassium chloride (KCl) and at least 0.01 mM calciumchloride (CaCl2). The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. in “in vivo” methods occurring liquids such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well, which are suitable foradministration to a person. The term “compatible” as used herein meansthat the constituents of the inventive vaccine are capable of beingmixed with the mRNA according to the invention as defined herein, insuch a manner that no interaction occurs, which would substantiallyreduce the pharmaceutical effectiveness of the inventive vaccine undertypical use conditions. Pharmaceutically acceptable carriers, fillersand diluents must, of course, have sufficiently high purity andsufficiently low toxicity to make them suitable for administration to aperson to be treated. Some examples of compounds which can be used aspharmaceutically acceptable carriers, fillers or constituents thereofare sugars, such as, for example, lactose, glucose, trehalose andsucrose; starches, such as, for example, corn starch or potato starch;dextrose; cellulose and its derivatives, such as, for example, sodiumcarboxymethylcellulose, ethylcellulose, cellulose acetate; powderedtragacanth; malt; gelatin; tallow; solid glidants, such as, for example,stearic acid, magnesium stearate; calcium sulfate; vegetable oils, suchas, for example, groundnut oil, cottonseed oil, sesame oil, olive oil,corn oil and oil from theobroma; polyols, such as, for example,polypropylene glycol, glycerol, sorbitol, mannitol and polyethyleneglycol; alginic acid.

The choice of a pharmaceutically acceptable carrier is determined, inprinciple, by the manner, in which the pharmaceutical composition orvaccine according to the invention is administered. The composition orvaccine can be administered, for example, systemically or locally.Routes for systemic administration in general include, for example,transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,composition or vaccines according to the present invention may beadministered by an intradermal, subcutaneous, or intramuscular route,preferably by injection, which may be needle-free and/or needleinjection. Compositions/vaccines are therefore preferably formulated inliquid or solid form. The suitable amount of the vaccine or compositionaccording to the invention to be administered can be determined byroutine experiments, e.g. by using animal models. Such models include,without implying any limitation, rabbit, sheep, mouse, rat, dog andnon-human primate models. Preferred unit dose forms for injectioninclude sterile solutions of water, physiological saline or mixturesthereof. The pH of such solutions should be adjusted to about 7.4.Suitable carriers for injection include hydrogels, devices forcontrolled or delayed release, polylactic acid and collagen matrices.Suitable pharmaceutically acceptable carriers for topical applicationinclude those which are suitable for use in lotions, creams, gels andthe like. If the inventive composition or vaccine is to be administeredperorally, tablets, capsules and the like are the preferred unit doseform. The pharmaceutically acceptable carriers for the preparation ofunit dose forms which can be used for oral administration are well knownin the prior art. The choice thereof will depend on secondaryconsiderations such as taste, casts and storability, which are notcritical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

The inventive vaccine or composition can additionally contain one ormore auxiliary substances in order to further increase theimmunogenicity. A synergistic action of the mRNA contained in theinventive composition and of an auxiliary substance, which may beoptionally be co-formulated (or separately formulated) with theinventive vaccine or composition as described above, is preferablyachieved thereby. Depending on the various types of auxiliarysubstances, various mechanisms may play a role in this respect. Forexample, compounds that permit the maturation of dendritic cells (DCs),for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a firstclass of suitable auxiliary substances. In general, it is possible touse as auxiliary substance any agent that influences the immune systemin the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, suchas GM-CFS, which allow an immune response produced by theimmune-stimulating adjuvant according to the invention to be enhancedand/or influenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that—additional to induction of the adaptive immuneresponse by the encoded at least one antigen—promote the innate immuneresponse, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF, G-CSF,M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH. Preferably,such immunogenicity increasing agents or compounds are providedseparately (not co-formulated with the inventive vaccine or composition)and administered individually.

Further additives which may be included in the inventive vaccine orcomposition are emulsifiers, such as, for example, Tween; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

The inventive vaccine or composition can also additionally contain anyfurther compound, which is known to be immune-stimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventive vaccineor composition in this context, may be CpG nucleic acids, in particularCO-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA(ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-strandedCpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpGnucleic acid is preferably in the form of CpG-RNA, more preferably inthe form of single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acidpreferably contains at least one or more (mitogenic) cytosine/guaninedinucleotide sequence(s) (CpG motif(s)). According to a first preferredalternative, at least one CpG motif contained in these sequences, thatis to say the C (cytosine) and the G (guanine) of the CpG motif, isunmethylated. All further cytosines or guanines optionally contained inthese sequences can be either methylated or unmethylated. According to afurther preferred alternative, however, the C (cytosine) and the G(guanine) of the CpG motif can also be present in methylated form.

Application and Medical Use:

According to one aspect of the present invention, the mRNA sequence, the(pharmaceutical) composition or the vaccine may be used according to theinvention (for the preparation of a medicament) for the treatment orprophylaxis of Norovirus infections or disorders related thereto.

In this context, also included in the present invention are methods oftreating or preventing Norovirus infections or disorders relatedthereto, preferably as defined herein, by administering to a subject inneed thereof a pharmaceutically effective amount of the mRNA sequence,the (pharmaceutical) composition or the vaccine according to theinvention. Such a method typically comprises an optional first step ofpreparing the mRNA sequence, the composition or the vaccine of thepresent invention, and a second step, comprising administering (apharmaceutically effective amount of) said composition or vaccine to apatient/subject in need thereof. A subject in need thereof willtypically be a mammal. In the context of the present invention, themammal is preferably selected from the group comprising, without beinglimited thereto, e.g. goat, cattle, swine, dog, cat, donkey, monkey,ape, a rodent such as a mouse, hamster, rabbit and, particularly, human.A subject in need thereof may also be a non-mammalian vertebrate, e.g. abird (chicken).

The invention also relates to the use of the mRNA sequence, thecomposition or the vaccine according to the invention, preferably foreliciting an immune response in a mammal, preferably for the treatmentor prophylaxis of Norovirus infections or a related condition as definedherein.

The present invention furthermore comprises the use of the mRNAsequence, the (pharmaceutical) composition or the vaccine according tothe invention as defined herein for modulating, preferably for inducingor enhancing, an immune response in a mammal as defined herein, morepreferably for preventing and/or treating Norovirus infections, or ofdiseases or disorders related thereto. In this context, support of thetreatment or prophylaxis of Norovirus infections may be any combinationof a conventional Norovirus therapy method such as therapy withantivirals such as neuraminidase inhibitors (e.g. oseltamivir andzanamivir) and M2 protein inhibitors (e.g. adamantane derivatives), anda therapy using the RNA or the pharmaceutical composition as definedherein. Support of the treatment or prophylaxis of Norovirus infectionsmay be also envisaged in any of the other embodiments defined herein.Accordingly, any use of the mRNA sequence, the (pharmaceutical)composition or the vaccine according to the invention in co-therapy withany other approach, preferably one or more of the above therapeuticapproaches, in particular in combination with antivirals is within thescope of the present invention.

For administration, preferably any of the administration routes may beused as defined herein. In particular, an administration route is used,which is suitable for treating or preventing an Norovirus infection asdefined herein or diseases or disorders related thereto, by inducing orenhancing an adaptive immune response on the basis of an antigen encodedby the mRNA sequence according to the invention. Administration of thecomposition and/or the vaccine according to the invention may then occurprior, concurrent and/or subsequent to administering another compositionand/or vaccine as defined herein, which may in addition contain anothermRNA sequence or combination of mRNA sequences encoding a differentantigen or combination of antigens, wherein each antigen encoded by themRNA sequence according to the invention is preferably suitable for thetreatment or prophylaxis of Norovirus infections and diseases ordisorders related thereto. In this context, a treatment as definedherein may also comprise the modulation of a disease associated toNorovirus infection and of diseases or disorders related thereto.

According to a preferred embodiment of this aspect of the invention, the(pharmaceutical) composition or the vaccine according to the inventionis administered by injection. Any suitable injection technique known inthe art may be employed. Preferably, the inventive composition isadministered by injection, preferably by needle-less injection, forexample by jet-injection.

In one embodiment, the inventive composition comprises at least one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve ormore mRNAs as defined herein, each of which is preferably injectedseparately, preferably by needle-less injection. Alternatively, theinventive composition comprises at least one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mRNAs,wherein the at least one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 or more mRNAs are administered,preferably by injection as defined herein, as a mixture.

The immunization protocol for the immunization of a subject against anantigen or a combination of at least two, three, four, five, six, seven,eight, nine, ten, eleven, twelve or more antigens as defined hereintypically comprises a series of single doses or dosages of the(pharmaceutical) composition or the vaccine according to the invention.A single dosage, as used herein, refers to the initial/first dose, asecond dose or any further doses, respectively, which are preferablyadministered in order to “boast” the immune reaction. In this context,each single dosage preferably comprises the administration of the sameantigen or the same combination of antigens as defined herein, whereinthe interval between the administration of two single dosages can varyfrom at least one day, preferably 2, 3, 4, 5, 6 or 7 days, to at leastone week, preferably 2, 3, 4, 5, 6, 7 or 8 weeks. The intervals betweensingle dosages may be constant or vary over the course of theimmunization protocol, e.g. the intervals may be shorter in thebeginning and longer towards the end of the protocol. Depending on thetotal number of single dosages and the interval between single dosages,the immunization protocol may extend over a period of time, whichpreferably lasts at least one week, more preferably several weeks (e.g.2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks), even mare preferablyseveral months (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months).Each single dosage preferably encompasses the administration of anantigen, preferably of a combination of at least two, three, four, five,six, seven, eight, nine, ten, eleven, twelve or more antigens as definedherein and may therefore involve at least one, preferably 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 injections. In some cases, the composition orthe vaccine according to the invention is administered as a singledosage typically in one injection. In the case, where the vaccineaccording to the invention comprises separate mRNA formulations encodingdistinct antigens as defined herein, the minimum number of injectionscarried out during the administration of a single dosage corresponds tothe number of separate components of the vaccine. In certainembodiments, the administration of a single dosage may encompass morethan one injection for each component of the vaccine (e.g. a specificmRNA formulation comprising an mRNA encoding, for instance, oneantigenic peptide or protein as defined herein). For example, parts ofthe total volume of an individual component of the vaccine may beinjected into different body parts, thus involving more than oneinjection. In a more specific example, a single dosage of a vaccinecomprising four separate mRNA formulations, each of which isadministered in two different body parts, comprises eight injections.Typically, a single dosage comprises all injections required toadminister all components of the vaccine, wherein a single component maybe involve more than one injection as outlined above. In the case, wherethe administration of a single dosage of the vaccine according to theinvention encompasses more than one injection, the injection are carriedout essentially simultaneously or concurrently, i.e. typically in atime-staggered fashion within the time-frame that is required for thepractitioner to carry out the single injection steps, one after theother. The administration of a single dosage therefore preferablyextends over a time period of several minutes, e.g. 2, 3, 4, 5, 10, 15,30 or BD minutes.

Administration of the mRNA sequence as defined herein, the(pharmaceutical) composition or the vaccine according to the inventionmay be carried out in a time staggered treatment. A time staggeredtreatment may be e.g. administration of the mRNA sequence, thecomposition or the vaccine prior, concurrent and/or subsequent to aconventional therapy of Norovirus infections or diseases or disordersrelated thereto, e.g. by administration of the mRNA sequence, thecomposition or the vaccine prior, concurrent and/or subsequent to atherapy or an administration of a therapeutic suitable for the treatmentor prophylaxis of Norovirus infections or diseases or disorders relatedthereto. Such time staggered treatment may be carried out using e.g. akit, preferably a kit of parts as defined herein.

Time staggered treatment may additionally or alternatively also comprisean administration of the mRNA sequence as defined herein, the(pharmaceutical) composition or the vaccine according to the inventionin a form, wherein the mRNA encoding an antigenic peptide or protein asdefined herein or a fragment or variant thereof, preferably forming partof the composition or the vaccine, is administered parallel, prior orsubsequent to another mRNA sequence encoding an antigenic peptide orprotein as defined above, preferably forming part of the same inventivecomposition or vaccine. Preferably, the administration (of all mRNAsequences) occurs within an hour, more preferably within 30 minutes,even more preferably within 15, 10, 5, 4, 3, or 2 minutes or even within1 minute. Such time staggered treatment may be carried out using e.g. akit, preferably a kit of parts as defined herein.

In a preferred embodiment, the pharmaceutical composition or the vaccineof the present invention is administered repeatedly, wherein eachadministration preferably comprises individual administration of the atleast one mRNA of the inventive composition or vaccine. At each timepoint of administration, the at least one mRNA may be administered morethan once (e.g. 2 or 3 times). In a particularly preferred embodiment ofthe invention, at least two, three, four, five, six or more mRNAsequences (each encoding a distinct one of the antigens as definedherein) are administered at each time point, wherein each mRNA isadministered twice by injection, distributed over the four limbs.

Kit or Kit of Parts:

According to another aspect of the present invention, the presentinvention also provides a kit, in particular a kit of parts, comprisingthe mRNA sequence as defined herein, the (pharmaceutical) composition,and/or the vaccine according to the invention, optionally a liquidvehicle for solubilising and optionally technical instructions withinformation on the administration and dosage of the mRNA sequence, thecomposition and/or the vaccine. The technical instructions may containinformation about administration and dosage of the mRNA sequence, thecomposition and/or the vaccine. Such kits, preferably kits of parts, maybe applied e.g. for any of the above mentioned applications or uses,preferably for the use of the mRNA sequence according to the invention(for the preparation of an inventive medicament, preferably a vaccine)for the treatment or prophylaxis of Norovirus infections or diseases ordisorders related thereto. The kits may also be applied for the use ofthe mRNA sequence, the composition or the vaccine as defined herein (forthe preparation of an inventive vaccine) for the treatment orprophylaxis of Norovirus infections or diseases or disorders relatedthereto, wherein the mRNA sequence, the composition and/or the vaccinemay be capable of inducing or enhancing an immune response in a mammalas defined above. Such kits may further be applied for the use of themRNA sequence, the composition or the vaccine as defined herein (for thepreparation of an inventive vaccine) for modulating, preferably foreliciting, e.g. to induce or enhance, an immune response in a mammal asdefined above, and preferably for supporting treatment or prophylaxis ofNorovirus infections or diseases or disorders related thereto. Kits ofparts, as a special form of kits, may contain one or more identical ordifferent compositions and/or one or more identical or differentvaccines as described herein in different parts of the kit. Kits ofparts may also contain an (e.g. one) composition, an (e.g. one) vaccineand/or the mRNA sequence according to the invention in different partsof the kit, e.g. each part of the kit containing an mRNA sequence asdefined herein, preferably encoding a distinct antigen. Preferably, thekit or the kit of parts contains as a part a vehicle for solubilisingthe mRNA according to the invention, the vehicle preferably beingRinger-lactate solution. Any of the above kits may be used in atreatment or prophylaxis as defined above.

In another embodiment of this aspect, the kit according to the presentinvention may additionally contain at least one adjuvant. In a furtherembodiment, the kit according to the present invention may additionallycontain at least one further pharmaceutically active component,preferably a therapeutic compound suitable for treatment and/orprophylaxis of cancer or a related disorder. Moreover, in anotherembodiment, the kit may additionally contain parts and/or devicesnecessary or suitable for the administration of the composition or thevaccine according to the invention, including needles, applicators,patches, injection-devices.

Preferred Items:

In one embodiment the invention relates to subject matter summarized asfollows:

Item 1. Artificial nucleic acid comprising at least one coding regionencoding at least one polypeptide derived from a Norovirus, and/or afragment or variant thereof.

Item 2. The artificial nucleic acid according to item 1, wherein the atleast one encoded polypeptide is selected from the group consisting of anon-structural protein derived from a Norovirus and/or a capsid proteinderived from a Norovirus, and/or a fragment or variant thereof.

Item 3. The artificial nucleic acid according to item 1 or 2, whereinthe at least one encoded polypeptide is selected from the groupconsisting of Norovirus non-structural proteins NS1/NS2, NS3, NS4, NS5,NS6, NS7, Norovirus capsid protein VP1 and Norovirus capsid protein VP2,and/or a fragment or variant thereof.

Item 4. The artificial nucleic acid according to any one of items 1 to3, wherein the artificial nucleic acid is derived from a Norovirusselected from the group consisting of genogroup I Norovirus, genogroupII Norovirus, genogroup III Norovirus, genogroup IV Norovirus, andgenogroup V Norovirus; preferably the artificial nucleic acid is derivedfrom a Norovirus selected from the group consisting of a GI.1 to GI.17Norovirus, GII.1 to GII.24 Norovirus, GIII.1 to GIII.4 Norovirus, GIV.1to GIV.4 Norovirus and GV.1 to GV.4 Norovirus; more preferably, theartificial nucleic acid is derived from a Norovirus selected from thegroup consisting of GI.1 Norovirus and GII.4 Norovirus, even morepreferably, the artificial nucleic acid is derived from a GII.4Norovirus, still more preferably, the artificial nucleic acid is derivedfrom a GII.4 CIN-1 Norovirus or a GII.4 Sydney Norovirus or a GII.4Sydney 2012 Norovirus.

Item 5. The artificial nucleic acid according to any one of items 1 to4, wherein the at least one encoded polypeptide comprises at least oneNorovirus capsid protein VP1 or Norovirus capsid protein VP2 and/or afragment or a variant thereof.

Item 6. The artificial nucleic acid according to any one of items 1 to5, wherein the at least one encoded polypeptide comprises at least oneNorovirus capsid protein VP1 and/or a fragment or variant thereof.

Item 7. The artificial nucleic acid according to any one of items 1 to6, wherein the at least one encoded polypeptide comprises

-   -   (i) at least one of the amino acid sequences according to any        one of SEQ ID NOs: 1-4410; and/or    -   (ii) at least one of the amino acid sequences having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 50%, 57%, 58%, 59%, 60%, 51%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        amino acid sequence represented by any one of SEQ ID NOs:        1-4410; and/or    -   (iii) an orthologue or a paralogue of any one of SEQ ID NOs:        1-39090, 39713-39746; and/or a fragment or variant of any of        these sequences.

Item 8. The artificial nucleic acid according to any one of items 1 to7, wherein the at least one coding region comprises

-   -   (i) at least one of the nucleic acid sequences according to any        one of SEQ ID NOs: 4411-39590, 39713-39745; and/or    -   (ii) at least one of the nucleic acid sequences having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 00%, 61%, 62%, 63%, 64%, 65%, GA,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 75%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        nucleic acid sequence represented by any one of SEQ ID NOs:        4411-39690, 39713-39746; and/or    -   (iii) at least one complement of the nucleic acid sequences        which are capable of hybridizing with a nucleic acid sequence        comprising a sequence as shown in SEQ ID NOs: 4411-39590,        39713-39746, and/or to a nucleic acid encoding a polypeptide        having a sequence as shown in SEQ ID NOs: 1-4410: and/or    -   (iv) an orthologue or a paralogue of any one of SEQ ID NOs:        1-39690, 39713-39746; and/or a fragment or variant of any of        these sequences.

Item 9. The artificial nucleic acid according to any one of items 1 to8, wherein the artificial nucleic acid is monocistronic, bicistronic ormulticistronic.

Item 10. The artificial nucleic acid according to any one of items 1 to9, wherein the artificial nucleic acid is monocistronic and wherein thecoding region encodes a polypeptide comprising at least two differentNorovirus proteins as defined in any one of items 1 to 9, or a fragmentor variant thereof.

Item 11. The artificial nucleic acid according to any one of items 1 to9, wherein the artificial nucleic acid is bi- or multicistronic andcomprises at least two coding regions, wherein the at least two codingregions encode at least two polypeptides, wherein each of the at leasttwo polypeptides comprises at least one Norovirus protein as defined inany one of items 1 to 9, or a fragment or variant of any one of theseproteins, wherein the at least two polypeptides are preferably differentpolypeptides.

Item 12. The artificial nucleic acid according to any one of items 1 to11, wherein the artificial nucleic acid is an RNA, preferably an mRNA.

Item 13. The artificial nucleic acid according to any one of items 1 to12, wherein the artificial nucleic acid comprises a 5′-cap structure.

Item 14. The artificial nucleic acid according to any one of items 1 to13, wherein the G/C content of the coding region of the mRNA sequence isincreased compared to the G/C content of the corresponding codingsequence of the wild type mRNA, or wherein the C content of the codingregion of the mRNA sequence is increased compared to the C content ofthe corresponding coding sequence of the wild type mRNA, or wherein thecodon usage in the coding region of the mRNA sequence is adapted to thehuman codon usage, or wherein the codon adaptation index (CAI) isincreased or maximised in the coding region of the mRNA sequence,wherein the encoded amino acid sequence of the mRNA sequence ispreferably not being modified compared to the encoded amino acidsequence of the wild type mRNA.

Item 15. The artificial nucleic acid according to any one of items 1 to14, wherein

-   -   (i) the at least one coding region comprises a nucleic acid        sequence, which is codon-optimized; and/or    -   (ii) wherein the at least one coding sequence comprises a        nucleic acid sequence, which is identical or at least 50%, BA,        70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, or 99% identical to a nucleic acid sequence        selected from the group consisting of SEQ ID NOs: 8821-13230,        26461-39690, 39715, 39716, 39717, 39720, 39721, 39724, 39725,        39728, 39729, 39730, 39733, 39734, 39737, 39738, 39741, 39742,        39745 and 39746, or a fragment or variant of any of these        sequences; and/or    -   (iii) wherein the at least one coding sequence comprises a        nucleic acid sequence, which is identical or at least 50%, 80%,        70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, or 99% identical to a nucleic acid sequence        selected from the group consisting of SEQ ID NOs: 13231-17840,        or a fragment or variant of any of these sequences; and/or    -   (iv) the artificial nucleic acid according to any one of the        preceding items, wherein the at least one coding sequence        comprises a nucleic acid sequence, which is identical or at        least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,        92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic        acid sequence selected from the group consisting of SEQ ID NOs:        17641-22050, or a fragment or variant of any of these sequences;        and/or    -   (v) the artificial nucleic acid according to any one of the        preceding items, wherein the at least one coding sequence        comprises a nucleic acid sequence, which is identical or at        least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,        92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic        acid sequence selected from the group consisting of SEQ ID NOs:        22051-26460, or a fragment or variant of any of these sequences.

Item 16. The artificial nucleic acid according to any one of items 1 to15, wherein the artificial nucleic acid comprises at least one histonestem-loop.

Item 17. The artificial nucleic acid according to item 16, wherein theat least one histone stem-loop comprises a nucleic acid sequenceaccording to the following formulae (I) or (II):

-   -   formula (I) (stem-loop sequence without stem bordering        elements):

-   -   formula (II) (stem-loop sequence with stem bordering elements):

-   -   wherein:    -   stem1 or stem2 bordering elements N₁₋₆ is a consecutive sequence        of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even        more preferably of 3 to 5, most preferably of 4 to 5 or 5 N,        wherein each N is independently from another selected from a        nucleotide selected from A, U, T, G and C, or a nucleotide        analogue thereof;    -   stem1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse        complementary with element stem2, and is a consecutive sequence        between of 5 to 7 nucleotides;        -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably            of 0 to 1, more preferably of 1 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof;        -   wherein N3-5 is a consecutive sequence of 3 to 5, preferably            of 4 to 5, more preferably of 4 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof, and        -   wherein G is guanosine or an analogue thereof, and may be            optionally replaced by a cytidine or an analogue thereof,            provided that its complementary nucleotide cytidine in stem2            is replaced by guanosine;        -   loop sequence [N₀₋₄(U/T)N_(0-4]) is located between elements            stem1 and stem2, and is a consecutive sequence of 3 to 5            nucleotides, more preferably of 4 nucleotides;        -   wherein each N₀₋₄ is independent from another a consecutive            sequence of 0 to 4, preferably of 1 to 3, more preferably of            1 to 2 N, wherein each N is independently from another            selected from a nucleotide selected from A, U, T, G and C or            a nucleotide analogue thereof; and        -   wherein U/T represents uridine, or optionally thymidine;    -   stem2 [N₃₋₅CN₀₋₂] is reverse complementary or partially reverse        complementary with element stem1, and is a consecutive sequence        between of 5 to 7 nucleotides;        -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably            of 4 to 5, more preferably of 4 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof;        -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably            of 0 to 1, more preferably of 1N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof; and        -   wherein C is cytidine or an analogue thereof, and may be            optionally replaced by a guanosine or an analogue thereof            provided that its complementary nucleotide guanosine in            stems is replaced by cytidine;    -   wherein    -   stem1 and stem2 are capable of base pairing with each other        forming a reverse complementary sequence, wherein base pairing        may occur between stem1 and stem2, or    -   forming a partially reverse complementary sequence, wherein an        incomplete base pairing may occur between stem1 and stem2.

Item 18. The artificial nucleic acid according to item 17, wherein theat least one histone stem-loop comprises a nucleic acid sequenceaccording to the following formulae (Ia) or (IIa):

-   -   formula (Ia) (stem-loop sequence without stem bordering        elements):

-   -   formula (IIa) (stem-loop sequence with stem bordering elements):

Item 19. The artificial nucleic acid according to any one of items 16 to18, wherein the at least one histone stem loop comprises a nucleic acidsequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710, or afragment or variant thereof.

Item 20. The artificial nucleic acid molecule according to any one ofitems 1 to 19, wherein the artificial nucleic acid comprises anuntranslated region (UTR).

Item 21. The artificial nucleic acid according to item 20, wherein theartificial nucleic acid comprises a 3′-UTR.

Item 22. The artificial nucleic acid according to item 21, wherein the3′-UTR comprises at least one heterologous 3′-UTR element.

Item 23. The artificial nucleic acid according to item 21 or 22, whereinthe 3′-UTR comprises a poly(A) sequence and/or a poly(C) sequence.

Item 24. The artificial nucleic acid according to item 23, wherein thepoly(A) sequence comprises 10 to 200, 10 to 100, 40 to 80 or 50 to 70adenosine nucleotides, and/or the poly(C) sequence comprises 10 to 200,10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides.

Item 25. The artificial nucleic acid according to any one of items 1 to24, wherein the at least one heterologous 3′-UTR element comprises anucleic acid sequence derived from a 3′-UTR of a gene, which preferablyencodes a stable mRNA, or from a homolog, a fragment or a variant ofsaid gene.

Item 26. The artificial nucleic acid according to any one of items 1 to25, wherein the at least one heterologous 3′-UTR element comprises anucleic acid sequence derived from a 3′-UTR of a gene selected from thegroup consisting of an albumin gene, an α-globin gene, a β-globin gene,a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alphagene, or from a homolog, a fragment or a variant thereof.

Item 27. The artificial nucleic acid according to any one of items 1 to26, wherein the at least one heterologous 3′-UTR element comprises anucleic acid sequence derived from a 3′-UTR of an α-globin gene,preferably comprising the corresponding RNA sequence of the nucleic acidsequence according to SEQ ID NO: 39701, or SEQ ID NO: 39702, a homolog,a fragment, or a variant thereof.

Item 28. The artificial nucleic acid according to any one of items 1 to27, wherein the at least one heterologous 3′-UTR element comprises anucleic acid sequence, which is derived from the 3′-UTR of a vertebratealbumin gene or from a variant thereof, preferably from the 3′-UTR of amammalian albumin gene or from a variant thereof, more preferably fromthe 3′-UTR of a human albumin gene or from a variant thereof, even morepreferably from the 3′-UTR of the human albumin gene according toGenbank Accession number NM_000477.5, or from a fragment or variantthereof.

Item 29. The artificial nucleic acid according to any one of items 1 to28, wherein the at least one heterologous 3′-UTR element comprises anucleic acid sequence according to any one of SEQ ID NO: 39703 to SEQ IDNO: 39708, or a homing, a fragment or a variant thereof.

Item 30. The artificial nucleic acid according to any one of items 1 to29, wherein the artificial nucleic acid comprises a 5′-UTR.

Item 31. The artificial nucleic acid sequence according to any one ofitems 1 to 3D, wherein the 5′-UTR comprises at least one heterologous5′-UTR element.

Item 32. The artificial nucleic acid according to any one of items 1 to31, wherein the at least one heterologous 5′-UTR element comprises anucleic acid sequence, which is derived from the 5′-UTR of a TDP gene,preferably from a corresponding RNA sequence, or a homolog, a fragment,or a variant thereof, preferably lacking the 5′TOP motif.

Item 33. The artificial nucleic acid according to any one of items 1 to32, wherein the at least one heterologous 5′-UTR element comprises anucleic acid sequence, which is derived from a 5′-UTR of a TDP geneencoding a ribosomal protein, preferably from a corresponding RNAsequence, or from a homolog, a fragment or a variant thereof, preferablylacking the 5′TOP motif.

Item 34. The artificial nucleic acid according to any one of items 1 to33, wherein the at least one heterologous 5′-UTR element comprises anucleic acid sequence, which is derived from a 5′-UTR of a TOP geneencoding a ribosomal Large protein (RPL), preferably RPL32 or RPL35A, orfrom a gene selected from the group consisting of HSD17B4, ATP5A1, AIG1,ASAH1, COX6C or ABCB7 (MDR), or from a homolog, a fragment or variant ofany one of these genes, preferably lacking the 5′TOP motif.

Item 35. The artificial nucleic acid according to any one of items 1 to34, wherein the at least one heterologous 5′-UTR element comprises anucleic acid sequence according to SEQ ID NO: 39691 to SEQ ID NO: 39694,or a homolog, a fragment or a variant thereof.

Item 36. The artificial nucleic acid according to any one of items 1 to35 comprising, preferably in 5′ to 3′ direction, the following elements:

-   -   a) optionally a 5′-cap structure, preferably m7GpppN,    -   b) a coding region encoding at least one polypeptide derived        from a Norovirus as described herein, preferably VP1, or a        fragment or variant thereof,    -   c) optionally a poly(A) tail, preferably consisting of 10 to        200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides,    -   d) optionally a poly(C) tail, preferably consisting of 10 to        200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine        nucleotides, and    -   e) optionally a histone stem-loop, preferably comprising the RNA        sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.

Item 37. The artificial nucleic acid according to any one of items 1 to36 comprising, preferably in 5′ to 3′ direction, the following elements:

-   -   a) optionally a 5′-cap structure, preferably m7GpppN,    -   b) a coding region encoding at least one polypeptide derived        from a Norovirus, preferably VP1 as described herein, or a        fragment or variant thereof,    -   c) a 3′-UTR element comprising a nucleic acid sequence, which is        derived from an α-globin gene, preferably comprising the        corresponding RNA sequence of the nucleic acid sequence        according to SEQ ID NO: 39701, or SEQ ID NO: 39702, or a        homolog, a fragment or a variant thereof,    -   d) optionally a poly(A) tail, preferably consisting of 10 to        200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides,    -   e) optionally a poly(C) tail, preferably consisting of 10 to        200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine        nucleotides, and    -   f) optionally a histone stem-loop, preferably comprising the RNA        sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.

Item 38. The artificial nucleic acid according to any one of items 1 to37, wherein the artificial nucleic acid comprises a nucleic acidsequence according to any one of SEQ ID NO: 39713-39749, preferably anucleic acid sequence according to any one of SEQ ID NO: 39719, 39721,39729, 39734, 39738, 39725, or a fragment or variant of any of thesesequences.

Item 39. The artificial nucleic acid according to any one of items 1 to38, comprising, preferably in 5′ to 3′ direction, the followingelements:

-   -   a) optionally a 5′-cap structure, preferably m7GpppN,    -   b) a 5′-UTR element, which comprises or consists of a nucleic        acid sequence, which is derived from the 5′-UTR of a TOP gene,        preferably comprising a nucleic acid sequence according to SEQ        ID NO: 39991, or SEQ ID NO: 39692, or a homolog, a fragment or a        variant thereof,    -   c) a coding region encoding at least one polypeptide derived        from a Norovirus, preferably VP1 as described herein, or a        fragment or variant thereof,    -   d) a 3′-UTR element comprising a nucleic acid sequence, which is        derived from an albumin gene, preferably comprising the        corresponding RNA sequence of the nucleic acid sequence        according to SEQ ID NO: 39705, or SEQ ID NO: 39706, or a        homolog, a fragment or a variant thereof,    -   e) optionally a poly(A) tail, preferably consisting of 10 to        200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides,    -   f) optionally a poly(C) tail, preferably consisting of 10 to        200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine        nucleotides, and    -   g) optionally a histone stem-loop, preferably comprising the RNA        sequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710.

Item 40. The artificial nucleic acid according to any one of items 1 to39, wherein the artificial nucleic acid comprises a nucleic acidsequence according to any one of SEQ ID NOs: 39713-39746, preferably anucleic acid sequence according to any one of SEQ ID NOs: 39716, 39721,39729, 39734, 39738, 39725, or a fragment or variant of any of thesesequences.

Item 41. The artificial nucleic acid according to any one of items 1 to40, wherein the coding region comprises a modified nucleic acidsequence.

Item 42. The artificial nucleic acid according to any one of items 1 to41, wherein the at least one coding region comprises a nucleic acidsequence encoding a molecular tag and wherein the molecular tag isselected from the group consisting of a FLAG tag, aglutathione-S-transferase (GST) tag, a His tag, a Myc tag, an E tag, aStrep tag, a green fluorescent protein (GFP) tag and an HA tag.

Item 43. Composition comprising at least one artificial nucleic acid asdefined by any one of items 1 to 42 and a pharmaceutically acceptablecarrier.

Item 44. The composition according to item 43, wherein the at least onemRNA is complexed with one or more cationic or polycationic compounds,preferably with cationic or polycationic polymers, cationic orpolycationic peptides or proteins, e.g. protamine, cationic orpolycationic polysaccharides and/or cationic or polycationic lipids.

Item 45. The composition according to any one of items 43 to 44, whereinthe N/P ratio of the at least one mRNA to the one or more cationic orpolycationic compounds is in the range of about 0.1 to 20, including arange of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and ofabout 0.7 to 1.5.

Item 46. The composition according to any one of items 43 to 45comprising the at least one mRNA, which is complexed with one or marecationic or polycationic compounds, and at least one free mRNA.

Item 47. The composition according to any one of items 43 to 46, whereinthe at least one complexed mRNA is identical to the at least one freemRNA.

Item 48. The composition according to any one of items 43 to 47, whereinthe mRNA is complexed with one or more lipids, thereby formingliposomes, lipid nanoparticles and/or lipoplexes.

Item 49. The composition according to any one of items 43 to 48, whereinthe composition comprises at least one adjuvant.

Item 50. The composition according to any one of items 43 to 49, wherein

-   -   a) the composition comprises a plurality or more than one of the        mRNA sequences each defined in any one of items 1 to 42;    -   or    -   b) the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,        26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,        42, 43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic        acids as defined by any one of items 1 to 42, wherein each of        the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,        33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,        49, 50 or more artificial nucleic acids comprises at least one        coding region encoding at least one polypeptide comprising a        Norovirus protein as defined in any one of items 1 to 42, and/or        a fragment or a variant of any one of these proteins, wherein        each coding region preferably encodes a different Norovirus        protein, more preferably each coding region encodes a capsid        protein, preferably VP1 of a different Norovirus.

Item 51. The composition according to any one of items 43 to 50, wherein

-   -   a) wherein each of the mRNA sequences encodes at least one        different antigenic peptide or protein derived from proteins of        the same Norovirus; and/or    -   b) the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,        26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,        42, 43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic        acids as defined by any one of items 1 to 42, wherein each of        the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,        33, 34, 35, 38, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,        49, 50 or more artificial nucleic acids comprises at least one        coding region encoding at least one polypeptide comprising at        least two different Norovirus proteins, preferably VP1 and VP2,        as defined in any one of items 1 to 42, and/or a fragment or a        variant of any one of these proteins.

Item 52. The composition according to any one of items 43 to 51, whereinthe at least one artificial nucleic acid is complexed at least partiallywith a cationic or polycationic compound and/or a polymeric carrier,preferably a cationic protein or peptide.

Item 53. The composition according to any one of items 43 to 52, wherein

-   -   (i) the ratio of complexed nucleic acid to free nucleic acid is        selected from a range of about 5:1 (w/w) to about 1:10 (w/w),        more preferably from a range of about 4:1 (w/w) to about 1:8        (w/w), even more preferably from a range of about 3:1 (w/w) to        about 1:5 (w/w) or 1:3 (w/w), wherein the ratio is most        preferably about 1:1 (w/w); or (ii) the mRNA is complexed with        one or more cationic or polycationic compounds in a weight ratio        selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w),        more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even        more preferably of about 4:1 (w/w) to about 1:1 (w:w) or of        about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio        of about 3:1 (w/w) to about 2:1 (w/w) of mRNA to cationic or        polycationic compound and/or with a polymeric carrier; or        optionally in a nitrogen/phosphate ratio of mRNA to cationic or        polycationic compound and/or polymeric carrier in the range of        about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and        most preferably in a range of about 0.5-1 or 0.7-1, and even        most preferably in a range of about 0.3-0.9 or 0.5-0.9;    -   and/or wherein the at least one artificial nucleic acid or mRNA        is complexed with one or more cationic or polycationic        compounds, preferably with cationic or polycationic polymers,        cationic or polycationic peptides or proteins, e.g. protamine,        cationic or polycationic polysaccharides and/or cationic or        polycationic lipids    -   and/or wherein the at least one artificial nucleic acid or mRNA        is complexed with one or more lipids and thereby forming        liposomes, lipid nanoparticles and/or lipoplexes.

Item 54. The composition according to any one of items 43 to 53 whereinthe composition comprises

-   -   (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more artificial        nucleic acids as defined in items 1 to 42; or    -   (ii) at least 10, 15, 20 or 50 artificial nucleic acids as        defined in items 1 to 42; or    -   (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificial        nucleic acids as defined in items 1 to 42;    -   and a pharmaceutically acceptable carrier, wherein preferably        the artificial nucleic acid encodes a capsid protein VP1 derived        from a Norovirus.

Item 55. The composition according to any one of items 43 to 54, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single GII        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or mare different 011 Noroviruses; or    -   (iii) the artificial nucleic acids are derived from a single        GIII Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50 or more different GIII Noroviruses; or    -   (iv) the artificial nucleic acids are derived from a single G1V        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GIV Noroviruses; or    -   (v) the artificial nucleic acids are derived from a single GU        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GV Noroviruses; or    -   (vi) the artificial nucleic acids are derived from a single GI        Norovirus and additionally from a single GII Norovirus, GIII        Norovirus, DIV Norovirus and/or GV Norovirus; or    -   (vii) the artificial nucleic acids are derived from a single GI        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI Noroviruses and additionally        from a single GII, GIII, GIV or GV Norovirus and/or from 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,        37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and/or        more GII, GIII, GIV or GV Noroviruses;    -   wherein preferably the artificial nucleic acids encode a capsid        protein VP1 derived from a Norovirus.

Item 56. The composition according to any one of items 43 to 55, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 49,        47, 48, 49, 50 or more different GI.1 Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single        GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50 or more different GII.4 Noroviruses; or    -   (iii) the artificial nucleic acids are derived from a single        GI.1 Norovirus and additionally from a single GII.4 Norovirus;        or    -   (iv) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI.1 Noroviruses and        additionally from a single GII.4 Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more GII.4        Noroviruses; and/or    -   wherein    -   (i) at least one of the nucleic acid sequences according to any        one of SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or    -   (ii) at least one of the nucleic acid sequences having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 90%, 91%, 62%, 63%, 64%, 65%, 69%,        67%, 98%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 89%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        nucleic acid sequence represented by any one of SEQ ID NO: 39713        to SEQ ID NO: 39749; and/or    -   (iii) at least one complement of the nucleic acid sequences        which are capable of hybridizing with a nucleic acid sequence        comprising a sequence as shown in SEQ ID NO: 39713 to SEQ ID NO:        39746; and/or    -   (iv) an orthologue or a paralogue of any one of SEQ ID NO: 39713        to SEQ ID NO: 39746; and/or a fragment or variant of any of        these sequences.

Item 57. Polypeptide encoded by the artificial nucleic acid according toany one of items 1 to 42.

Item 58. Polypeptide according to any one of items 1 to 42 comprising atleast one protein selected from the group consisting of NS1/NS2, NS3,NS4, NS5, NS6, NS7, VP1, and VP2 derived from Norovirus, or a fragmentor variant of any of these proteins, and at least one amino acidsequence selected from the group consisting of:

-   -   a) an amino acid sequence derived from a C-terminal fragment        from mature Norovirus capsid protein VP1, or a variant thereof,        wherein the C-terminal fragment consists of 3 to 20 amino acid        residues,    -   b) an amino acid sequence derived from a signal sequence of        Norovirus capsid protein VP1, or a fragment or variant thereof,        and    -   c) an amino acid sequence derived from an N-terminal fragment        from mature Norovirus non-structural protein NS1/NS2, NS3, NS4,        NS5, NS6, or NS7, or a variant thereof, wherein the N-terminal        fragment consists of 3 to 20 amino acid residues.

Item 59. The polypeptide according to any one of items 57 to 58comprising a molecular tag, wherein the molecular tag is selected fromthe group consisting of a FLAG tag, a glutathione-S-transferase (GST)tag, a His tag, a Myc tag, an E tag, a Strep tag, a green fluorescentprotein (GFP) tag and an HA tag.

Item 60. Composition comprising the polypeptide according to any one ofitems 57 to 59, and a pharmaceutically acceptable carrier.

Item 61. Vaccine comprising the artificial nucleic acid according to anyone of items 1 to 42, the composition according to any one of items 43to 56, the polypeptide according to any one of items 57 to 59, and/orthe composition according to item 60.

Item 62. The vaccine according to item 91, wherein the artificialnucleic acid according to any one of items 1 to 42, the compositionaccording to any one of items 43 to 56, the polypeptide according to anyone of items 57 to 59, or the composition according to item BD elicitsan adaptive immune response.

Item 63. The vaccine according to item 91 to 62, wherein the vaccinefurther comprises a pharmaceutically acceptable carrier.

Item 64. The vaccine according to any one of items 61 to 63 furthercomprising an adjuvant.

Item 65. The vaccine according to any one of items GI to 64, wherein thevaccine is multivalent and comprises

-   -   (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more artificial        nucleic acids as defined in items 1 to 42; or    -   (ii) at least 10, 15, 20 or 50 artificial nucleic acids as        defined in items 1 to 42; or    -   (iii) 2-10, 10-15, 15-2E1, 20-50, 50-100 or 100-200 artificial        nucleic acids as defined in items 1 to 42.

Item 66. The vaccine according to any one of items 61 to 65, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single 611        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 39, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GII Noroviruses; or    -   (iii) the artificial nucleic acids are derived from a single        GIII Norovirus or from 2, 3, 4, 5, ft 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50 or more different GIII Noroviruses; or    -   (iv) the artificial nucleic acids are derived from a single GIV        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GIV Noroviruses; or    -   (v) the artificial nucleic acids are derived from a single GV        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GV Noroviruses; or    -   (vi) the artificial nucleic acids are derived from a single GI        Norovirus and additionally from a single GII Norovirus, GIII        Norovirus, GIV Norovirus and/or GV Norovirus; or    -   (vii) the artificial nucleic acids are derived from a single GI        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI Noroviruses and additionally        from a single GII, GIII, GIV and/or GV Norovirus or from 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,        37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more        GII, GIII, GIV and/or GV Noroviruses.

Item 67. The vaccine according to any one of items GI to 66, wherein

-   -   (i) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI.1 Noroviruses; or    -   (ii) the artificial nucleic acids are derived from a single        GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50 or more different GII.4 Noroviruses; or    -   (iii) the artificial nucleic acids are derived from a single        GI.1 Norovirus and additionally from a single GII.4 Norovirus;        or    -   (iv) the artificial nucleic acids are derived from a single GI.1        Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,        47, 48, 49, 50 or more different GI.1 Noroviruses and        additionally from a single GII.4 Norovirus or from 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more GII.4        Noroviruses.

Item 68. Kit or kit of parts comprising the artificial nucleic acidaccording to any one of items 1 to 42, the composition according to anyone of items 43 to 56, the polypeptide according to any one of items 57to 59, the composition according to item 60 or the vaccine according toany one of items 61 to 67, optionally comprising a liquid vehicle forsolubilising, and optionally technical instructions providinginformation on administration and dosage of the components.

Item 69. The kit or kit of parts according to item 68 comprising Ringerlactate solution.

Item 70. The artificial nucleic acid according to any one of items 1 to42, the composition according to any one of items 43 to 56, thepolypeptide according to any one of items 57 to 59, the compositionaccording to item 60, the vaccine according to any one of items 61 to67, or the kit or kit of parts according to item 68 to 69 for use as amedicament.

Item 71. The artificial nucleic acid according to any one of items 1 to42, the composition according to any one of items 43 to 56, thepolypeptide according to any one of items 57 to 59, the compositionaccording to item BO, the vaccine according to any one of items GI to67, or the kit or kit of parts according to item 68 to 69 for use in thetreatment or prophylaxis of an infection with Norovirus or a disorderrelated to an infection with Norovirus.

Item 72. The artificial nucleic acid according to any one of accordingto any one of items 1 to 42, the composition according to any one ofitems 43 to 56, the polypeptide according to any one of items 57 to 59,the composition according to item 60, the vaccine according to any oneof items 61 to 67, or the kit or kit of parts according to item 68 to69, wherein the artificial nucleic acid, the composition, the vaccine orthe active component of the kit or kit of parts is administered byinjection, preferably by needle-less injection, more preferably by jetinjection.

Item 73. The artificial nucleic acid according to any one of items 1 to42, the composition according to any one of items 43 to 56, thepolypeptide according to any one of items 57 to 59, the compositionaccording to item 60, the vaccine according to any one of items 61 to67, or the kit or kit of parts according to item 68 to 69 for useaccording to any one of items 70 to 72, wherein the treatment orprophylaxis comprises the administration of a further activepharmaceutical ingredient.

Item 74. Method of treating or preventing a disorder, wherein the methodcomprises administering to a subject in need thereof the artificialnucleic acid according to any one of items 1 to 42, the compositionaccording to any one of items 43 to 56, the polypeptide according to anyone of items 57 to 59, the composition according to item 60, the vaccineaccording to any one of items 61 to 67, or the kit or kit of partsaccording to item 68 to 69.

Item 75. The method according to item 74, wherein the disorder is aninfection with Norovirus or a disorder related to an infection withNorovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows that transfection of HeLa cells with mRNAs coding forNorovirus antigen VP1 leads to the expression of the encoded protein.For cell transfection, mRNA constructs (construct ID R2) and (constructID R4) were used. Norovirus VP1 proteins were stained intracellularlywith a specific anti-Norovirus GII.4 antibody and a FITC labelledsecondary antibody and analyzed by FACS. A detailed description of theexperiment is provided in the examples section, Example 2.

FIG. 2: shows that transfection of HeLa cells with mRNAs coding forNorovirus antigen WI leads to the expression of the encoded protein. Forcell transfection, mRNA constructs (construct ID R26), (construct IDR27), and (construct ID R28) were used. Norovirus VP1 proteins werestained with a specific anti-Norovirus GII.4 antibody and a FITClabelled secondary antibody and analyzed by FACS. A detailed descriptionof the experiment is provided in the examples section, Example 2.

FIG. 3: shows that transfection of HeLa cells with mRNAs coding forNorovirus antigen WI leads to protein expression. For cell transfection,mRNA constructs (construct ID R2) and (construct ID R4) were used.Western blot analysis was performed cell lysates of transfected cells.As a control, a commercial VLP preparation (Medigen; 59 kD) was used.Norovirus VP1 proteins were stained with a specific anti-Norovirus GII.4antibody. M=marker lane: I=mRNA construct R2; 2=mRNA construct R4; 3=WFIcontrol; 4=empty control; 5=commercial VLP control. A detaileddescription of the experiment is provided in the examples section,Example 3.

FIG. 4: shows that immunization of mice with formulated Norovirus mRNAvaccine (Norovirus GC-optimized VP1_X124V; construct ID R4; protamineformulated) induced binding IgG1 and IgG2 antibodies, both in ahomologous ELISA design (FIG. 4A; coating material VLP GII.4) and in aheterologous ELISA design (FIG. 4B; coating material VLP GII.4 2011).1=group vaccinated with Norovirus mRNA vaccine; 2=buffer control group.A detailed description of the experiment is provided in the examplessection, Example 4.1.

FIG. 5: shows that immunization of mice with formulated Norovirus mRNAvaccine (Norovirus GC-optimized VP1_X124V; construct ID R4; protamineformulated) induced heterologous blocking antibodies. The results of aHisto-Blood Group Antigen (HBGA) assay in serum dilution 1:12.5 areshown. I=group vaccinated with Norovirus mRNA vaccine; 2=buffer controlgroup. A detailed description of the experiment is provided in theexamples section, Example 4.2.

FIG. 6: shows that immunization of mice with formulated Norovirus mRNAvaccine (Norovirus GC-optimized VP1_X124V; construct ID R4; protamineformulated) induced antigen specific T-cell responses. The results of anICS assay are shown (CD8+ T-cells). 1=group vaccinated with NorovirusmRNA vaccine; 2=buffer control group. A detailed description of theexperiment is provided in the examples section, Example 4.3.

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

Example 1: Preparation of mRNA for In Vitro and In Vivo Experiments

1.1. Preparation of DNA and mRNA Constructs:

For the present examples, DNA sequences encoding Norovirus antigenicproteins, derived from three or more different Norovirus strains wereprepared and used for subsequent RNA in vitro transcription reactions.The prepared RNA constructs (coding sequences (cds) and mRNA sequences)are listed in Table 4 below.

Most DNA sequences were prepared by modifying the wild type encoding DNAsequences by introducing a codon modified sequence or GC-optimizedsequence for stabilization, using three or more different in silicoalgorithms that e.g. increase the GC content of the respective codingsequence (indicated as “GC opt 1”, “GC opt 2”, “GC opt 3”, “GC opt 4”,“opt 5”, “opt 6”, “opt 7” in Table 4; further details relating tosequence modifications are provided in the specifications of theinvention). Some DNA sequences were used as a wild type coding sequence,without altering the GC content and without altering the codon usage ofthe coding sequence (indicated as “wt” in Table 4).

DNA sequences were prepared by modifying the wild type encoding DNAsequences by introducing a GC-optimized sequence for stabilization,using an in silica algorithms that increase the GC content of therespective coding sequence (e.g., indicated as “opt1” in Table 4, seeexplanation in the paragraph above).

Moreover, sequences were introduced into a pUC19 derived vector andmodified to comprise stabilizing sequences derived fromalpha-globin-3′-UTR, a stretch of 30 cytosines, a histone-stem-loopstructure, and a stretch of 64 adenosines at the 3′-terminal end(poly-A-tail), indicated as “design 1” in Table 4. Other sequences wereintroduced into a pUC19 derived vector to comprise stabilizing sequencesderived from 32L4 5′-UTR ribosomal FOP UTR and 3′-UTR derived fromalbumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, anda stretch of 64 adenosines at the 3′-terminal end (poly-A-tail),indicated as “design 2” in Table 4. Further details are relating mRNAconstruct design are provided in the specifications of the invention)

The obtained plasmid DNA constructs were transformed and propagated inbacteria (Escherichia coli) using common protocols known in the art.

TABLE 4 VP1 coding sequences, protein sequences and mRNA constructsConstruct RNA ID description Norovirus strain RNA design SEQ ID NO R1mRNA VP1_(X124V) GII.4-031693-USA-2003 design 1, wt 39713 R2 mRNAVP1_(X124V) GII.4-031693-USA-2003 design 2, wt 39714 R3 mRNA VP1_(X124V)GII.4-031693-USA-2003 design 1, GC opt 1 39715 R4 mRNA VP1_(X124V)GII.4-031693-USA-2003; C1N1 design 2, GC opt 1 39716 R5 proteinVP1_(X124V) GII.4-031693-USA-2003 Protein* 2358 R6 cds VP1_(X124V)GII.4-031693-USA-2003 wild type, wt 6768 R7 cds VP1_(X124V)GII.4-031693-USA-2003 GC opt 1 39717 R8 cds VP1_(X124V)GII.4-031693-USA-2003 GC opt 2 11178 R9 cds VP1_(X124V)GII.4-031693-USA-2003 opt 5 15588 R10 cds VP1_(X124V)GII.4-031693-USA-2003 opt 6 19998 R11 cds VP1_(X124V)GII.4-031693-USA-2003 opt 7 24408 R12 cds VP1_(X124V)GII.4-031693-USA-2003 GC opt 3 28818 R13 cds VP1_(X124V)GII.4-031693-USA-2003 GC opt 4 33228 R14 mRNA Capsidprotein GII.4Farmington Hills-2002-USA design 1 39718 R15 mRNA Capsidprotein GII.4Farmington Hills-2002-USA design 2 39719 R16 mRNA Capsidprotein GII.4Farmington Hills-2002-USA design 1, GC opt 1 39720 R17 mRNACapsidprotein GII.4 Farmington Hills-2002-USA design 2, GC opt 1 39721R18 protein Capsidprotein GII.4 Farmington Hills-2002-USA Protein* 1487R19 cds Capsidprotein GII.4 Farmington Hills-2002-USA wild type 5897 R20cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 2 10307 R21 cdsCapsidprotein GII.4 Farmington Hills-2002-USA opt 5 10307 R22 cdsCapsidprotein GII.4 Farmington Hills-2002-USA opt 6 19127 R23 cdsCapsidprotein GII.4 Farmington Hills-2002-USA opt 7 23537 R24 cdsCapsidprotein GII.4 Farmington Hills-2002-USA GC opt 3 27947 R25 cdsCapsidprotein GII.4 Farmington Hills-2002-USA GC opt 4 32357 R26 mRNAVP1 GII.4-2006b 092895-USA-2008 design 2, GC opt 1 39729 R27 mRNA VP1GII.4-GZ2010-L87-Guangzhou-2011 design 2, GC opt 1 39734 R28 mRNA VP1GII.4-USA-1997 design 2, GC opt 1 39738 R29 mRNA VP1GI.1-USA-1968-Capsidprotein design 2, GC opt 1 39725 *protein sequenceis back translated into RNA according to the above paragraph “G/Ccontent modification”

1.2. RNA In Vitro Transcription:

The DNA plasmids prepared according to paragraph 1.1 were enzymaticallylinearized using EcoRI and transcribed in vitro using DNA dependent T7RNA polymerase in the presence of a nucleotide mixture and cap analog(m7GpppG) under suitable buffer conditions. The obtained mRNAs werepurified using PureMessenger® (CureVac, Tübingen, Germany: WO2008/077592 A1) and used for in vitro and in vivo experiments.

1.3. Preparation of Protamine Formulated RNA Vaccine:

The obtained mRNA, e.g. HPLC purified RNA, was complexed with protamineby addition of protamine-trehalose solution to RNA solution at aRNA:protamine weight to weight ratio of 2:1. Then, complexed RNA wasmixed with non-complexed RNA in a ratio of 50% free RNA and 50%complexed RNA to obtain formulated RNA. Formulated RNA was used for inviva vaccination experiments.

1.4. Preparation of LNP Formulated RNA Vaccine:

RNA is encapsulated in lipid nanoparticle (LNP) using establishedprotocols known in the art. Briefly, LNP-encapsulated RNA is preparedusing an ionizable amino lipid (cationic lipid), phospholipid,cholesterol and a PEGylated lipid. Cationic lipid, DSPC, cholesterol andPEG-lipid are solubilized in ethanol. RNA is diluted to a totalconcentration of about 0.05 mg/mL in 50 mM citrate buffer pH 4. Syringepumps are used to mix the ethanolic lipid solution with RNA at a ratioof about 1:6 to 1:2 (vol/vol). Ethanol is then removed and the externalbuffer replaced with PBS by dialysis. Lipid nanoparticles are filteredthrough a 0.2 μm pore sterile filter. Lipid nanoparticle particlediameter size may be determined by quasi-elastic light scattering usinga Malvern Zetasizer Nano (Malvern, UK).

Example 2: Expression of Norovirus VP1 Antigens in HeLa Cells andAnalysis by FACS

To determine in vitro protein expression of the inventive Norovirus mRNAconstructs, Hela cells were transfected with mRNA constructs encodingNorovirus VP1 antigens and analyzed by intracellular FACS staining. Forcell transfection, an mRNA comprising VP1_X124V (GII.4-031693-USA-2003)wild type coding sequence (SEQ ID NO: 39714; construct ID R2) an mRNAcomprising VP1_X124V (GII.4-031693-USA-2003) GC-optimized codingsequence (SEQ ID NO: 39716; construct ID R4), an mRNA comprising VP1(GII.4-2006b 092895-USA-2008) GC-optimized coding sequence (SEQ ID NO:39729; construct ID R26), an mRNA comprising VP1(GII.4-GZ2010-L87-Guangzhou-2011) GC-optimized coding sequence (SEQ IDNO: 39734; construct R27) and an mRNA comprising VP1NOV(GII.4-USA-1997)-Capsidprotein GC-optimized coding sequence (SEQ IDNO: 39738; construct ID R28) were used. The detailed description of theperformed experiment is provided below.

HeLa cells were seeded in a 6-well plate at a density of 400,000cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1%Pen/Strep), 24 h prior to transfection. Cells were transfected with 1 μgand 2 μg mRNA per construct using lipofectamine 2000 (Invitrogen) astransfection reagent. As a negative control, water for injection (WFI)was used.

24 hours post transfection, transfected HeLa cells were stained with acommercial mouse anti-Norovirus GII.4 antibody [2000-G5] (Abcam; 1:500)and an anti-mouse FITC labelled secondary antibody (F5262 from Sigma;1:500) after Cytofix/Cytoperm (BD Biosciences) treatment according tomanufacturer's protocol. Subsequently, cells were analyzed by flowcytometry (FACS) on a BD FACS Canto II using the FACS Diva software.Quantitative analysis of the fluorescent FITC signal was performed usingthe FlowJo software package (Tree Star, Inc.). The results of the FACSexpression analysis are shown in FIG. 1 and FIG. 2.

Results:

FIG. 1 and FIG. 2 show that the Norovirus proteins were expressed in Macells transfected with the mRNA constructs R2, R4, R26, R27 and R28.Overall, around 80%-90% of transfected cells showed positive FITCsignal, indicating that the inventive constructs tested here were ableto efficiently drive protein expression without affecting cellviability. Of note, the data suggests that analogous mRNA constructsencoding other Norovirus VP1 or VP2 antigens (as defined in thespecifications or listed in Table 1 and Table 3) may also drive proteinexpression in a similar manner.

Example 3: Analysis of Protein Expression Using Western Blot

To determine in vitro protein expression upon HeLa cell transfectionwith the inventive mRNA constructs, HeLa cells were transientlytransfected with an mRNA constructs comprising VP1_X124V codingsequences. Cell lysates were prepared and analyzed using western blot.The detailed description of the performed experiment is provided below.

HeLa cells are transfected with 2 μg mRNA comprising wild type VP1_X124Vcoding sequence (SEQ ID NO: 39714; construct ID R2) and 2 μg mRNAcomprising GC-optimized VP1_X124V coding sequence (SEQ ID NO: 39718;construct ID R4). As a negative control water for injection (WFI) wasused. After 24 hours post transfection lysis buffer was added to theculture to prepare cellular lysates. Cellular lysates as well as acommercial Norovirus virus like particle (VLP; obtained from Medigen)were reduced by heating the samples to 95° C. for 10 minute.Subsequently, samples were subjected to SDS-PAGE underdenaturating/reducing conditions followed by western blot detection. Forthe detection of Norovirus proteins, a commercial mouse anti-NorovirusGII.4 antibody [2002-85] (1:250; Abcam) was used as primary antibodyfollowed by secondary goat anti mouse antibody coupled to IRDye 800CW(1:10000; Licor Biosciences). The results of the experiment are shown inFIG. 3.

Results:

FIG. 3 shows that the Norovirus proteins were expressed in HeLa cellstransfected with the inventive mRNA constructs (SEQ ID NOs: 39714 and39716). Of note, the data suggests that analogous mRNA constructsencoding other Norovirus antigens (as defined in the specifications orlisted in Table 1 and Table 3) may also drive protein expression in asimilar manner.

Example 4: Immunization of Mice and Evaluation of Norovirus SpecificImmune Responses

Female BALB/c mice were immunized intradermally (i.d.) with protamineformulated mRNA vaccine (construct ID R4) with doses, application routesand vaccination schedules as indicated in Table 5. As a negativecontrol, one group of mice was injected with buffer (ringer lactate,RiLa). All animals were vaccinated on day 0, 21 and 35. Blood sampleswere collected on day 49 for the determination of binding antibodytiters (using a homologous and heterologous ELISA assay), blockingantibody titers (using a heterologous HGBA assay) and T-cell responses(intracellular cytokine assay). Detailed descriptions of the performedexperiments are provided below.

TABLE 5 Vaccination regimen (Example 4) Group No of mice Treatment DoseRoute/Volume Vaccination schedule 1 6 Norovirus GC-optimized VP1_X124V80 μg i.d. d 0, d 21, d 35 SEQ ID NO: 39716; R4 2 × 50 μl Protamineformulated 2 6 100% RiLa Control i.m. d 0, d 21, d 35 1 × 25 μl

4.1. Determination of Homologous and Heterologous Immune Responses byELISA:

ELISA was performed using synthetically produced norovirus Virus likeparticles (VLP) as coating material. For the analysis of homologousimmune responses, plates were coated with VLP of the same strain ofgenotype GII.4 (GII.4 CIN1). For the analysis of heterologous immuneresponses, plates were coated with VLPs of another strain of genotypeGII.4 (GII.4 2011). Coated plates were incubated using respective serumdilutions, and binding of specific antibodies to the Norovirus coatingmaterial was detected using biotinylated isotype specific anti-mouseantibodies followed by streptavidin-HRP (horse radish peroxidase) withABTS as substrate. Endpoint titers of antibodies were measured by ELISAon day 49 after three vaccinations (see Table 5). The results are shownin FIG. 4A (homologous responses) and FIG. 4B (heterologous responses).

4.2. Determination of Blocking Antibody Titers Using a HBGA BlockingAssay:

Respective sera (day 49 after three vaccinations) were pre-incubatedwith synthetic norovirus VLPs (VLP (GII.4 2011)) and subsequently addedto HBGA coated plates. VLP binding to Histo-Blood Group Antigen (HBGA)was detected by norovirus specific antibodies. In the presence offunctional blocking antibodies in serum of immunized animals, VLPbinding to HBGA was blocked which results in a reduction of the detectedantibody signal. The respective blocking index was calculated ascommonly known in the art. The results of the assay are shown in FIG. 5.

4.3. Determination of Specific CD8 T-Cell Responses Using ICS:

Splenocytes from vaccinated mice were isolated according to a standardprotocol known in the art. Briefly, isolated spleens were grindedthrough a cell strainer and washed in PBS/1% FBS followed by red bloodcell lysis. After an extensive washing step with PBS/1% FBS splenocyteswere seeded into 96-well plates (2×10⁶ cells per well). The cells werestimulated with ten Norovirus CD8 peptide epitopes (1 μg/ml of eachpeptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BDBiosciences) and anti-CD107α-PE-Cy7 antibody, after one hour at 37° C.After stimulation, cells were washed and stained and for staining ofintracellular cytokines Cytofix/Cytoperm reagent (BD Biosciences) wasused according to the manufacturer's instructions. The followingantibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200),TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450(1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100.Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cellswere acquired using a Canto II flow cytometer (Beckton Dickinson). Flowcytometry data was analyzed using FlowJo software package (Tree Star,Inc.). Results for CD8+ T-cells are shown in FIG. 6.

Results:

FIG. 4 shows that the tested Norovirus mRNA vaccine induced Norovirusspecific IgG1 and IgG2 antibody titers in immunized mice. Humoral immuneresponse was demonstrated in a homologous ELISA setting (see FIG. 4A) aswell as in a heterologous ELISA setting (see FIG. 4B). Of note, theobserved heterologous humoral immune response (against another strain ofgenotype GII.4) is of particular importance for a broad protectionagainst Norovirus infections, as GII.4 strains are fast-evolving whichis challenging in successful Norovirus vaccine development.

FIG. 5 shows that the tested Norovirus mRNA vaccine induced Norovirusspecific blocking antibody titers in immunized mice in a homologous andheterologous HGBA assay setup, showing that also functional antibodieswere induced after immunization of mice with the inventive NorovirusmRNA vaccine. Of note, the induction of functional blocking antibodiesalso against another strain of genotype GII.4 demonstrates that the usedmRNA Norovirus vaccine may also confer broad protection againstdifferent Norovirus strains of genotype GII.4.

FIG. 6 shows that the tested Norovirus mRNA vaccine stimulated a robustCD8+ IFN-γ/TNF-α and CD8+CD107a/IFN-γ in spleen of immunized mice.

Overall, the results of the immunization experiments in mice show thatthe inventive Norovirus mRNA vaccine induced a broad immune responseengaging both the humoral-secretory and cellular immunity effector arms.Notably, heterologous immune responses were also observed (ELISA, HGBA).The data suggests that analogous mRNA constructs encoding otherNorovirus antigens (as defined in the specifications or listed in Table1 or Table 3) may also induce board immune responses in a similarmanner.

Example 5: Immunization of Mice and Further Evaluation of HeterologousImmune Responses

Female BALB/c mice are immunized intradermally (i.d.) andintramuscularily (i.m.) with protamine formulated or LNP formulated mRNAvaccines with doses, application routes and vaccination schedules asindicated in Table 6. As a negative control, one group of mice wasinjected with buffer (ringer lactate). All animals were vaccinated onday 0, 21 and 35. Blood samples are collected on day 49 for thedetermination of binding antibody titers (using a homologous andheterologous ELISA assay), blocking antibody titers (using a homologousand heterologous HGBA assay). Detailed descriptions of the performedexperiments are provided below.

TABLE 6 Vaccination regimen of mice (Example 5) Group No. of miceTreatment Dose Route/Volume Vaccination schedule 1 6 NorovirusGC-optimized VP1 80 μg i.d. d 0, d 21, d 35 GII.4-USA-1997 2 × 50 μl SEQID NO: 39738; R28 Protamine formulated 2 6 Norovirus GC-optimized VP1 80μg i.d. d 0, d 21, d 35 GII.4-2006b 092895-USA-2008 2 × 50 μl SEQ ID NO:39729; R26 Protamine formulated 3 6 Norovirus GC-optimized VP1 80 μgi.d. d 0, d 21, d 35 GII.4-GZ2010-L87-Guangzhou-2011 2 × 50 μl SEQ IDNO: 39734; R27 Protamine formulated 4 6 Norovirus GC-optimized VP1_X124V80 μg i.d. d 0, d 21, d 35 SEQ ID NO: 39716; R4 2 × 50 μl Protamineformulated 5 6 Norovirus GC-optimized VP1 20 μg i.m. d 0, d 21, d 35GII.4-USA-1997 2 × 25 μl SEQ ID NO: 39738; R28 LNP formulated 6 6Norovirus GC-optimized VP1 20 μg i.m. d 0, d 21, d 35 GII.4-2006b092895-USA-2008 2 × 25 μl SEQ ID NO: 39729; R26 LNP formulated 7 6Norovirus GC-optimized VP1 20 μg i.m. d 0, d 21, d 35GII.4-GZ2010-L87-Guangzhou-2011 2 × 25 μl SEQ ID NO: 39734; R27 LNPformulated 8 6 Norovirus GC-optimized VP1_X124V 20 μg i.m. d 0, d 21, d35 SEQ ID NO: 39716; R4 2 × 25 μl LNP formulated 9 6 100% RiLa Controli.m. d 0, d 21, d 35 1 × 25 μl

5.1. Determination of Homologous and Heterologous Immune Responses byELISA:

ELISA is performed essentially as described in Example 4.1. Plates arecoated with VLP 011.4 CIN1 and VLP GII.4 2011 to determine homologousand heterologous immune responses.

5.2. Determination of Blocking Antibody Titers Using a Heterologous HBGABlocking Assay:

The HGBA assay is performed essentially as described in Example 4.2. Therespective blocking index are calculated as commonly known in the art toevaluate homologous and heterologous cross neutralizing capacities ofthe used mRNA vaccines.

5.3. Determination of Specific CD8 T-Cell Responses Using ICS:

Multifunctional CD8 T-cell responses are analyzed as described inExample 4.3.

Example 6: Norovirus mRNA Vaccine Challenge Study in Gnotobiotic Pigs

6.1 Immunization of Gnotobiotic Pigs:

Gnotobiotic pigs are derived by hysterectomy from near-term sows andmaintained in germ-free isolator units. Pigs are fed commercialultra-high-temperature-treated sterile food. All pigs are confirmed asseronegative for Norovirus and germ-free prior to immunizationexperiments. Gnotobiotic pigs are immunized with protamine formulated orLNP formulated mRNA vaccines (monovalent, bivalent, or tetravalent) withdoses, application routes and vaccination schedules as indicated inTable 7. Analysis of immune responses is performed essentially asdescribed in Example 4 (ELISA, HGBA, and ICS).

TABLE 7 Vaccination regimen of pigs (Example B) Group No. of pigsTreatment Dose/Route Vaccination schedule 1 6 Monovalent vaccine:protamine formulated 240 μg d 0, d 21 GII.4-2006b 092895-USA-2008 i.d.SEQ ID NO: 39729; R26 2 × 200 μl 2 6 Bivalent vaccine: protamineformulated 240 μg d 0, d 21 GI.1-USA-1968-Capsidprotein (total) SEQ IDNO: 39725; R29 + i.d. GII.4-GZ2010-L87-Guangzhou-2011 2 × 200 μl SEQ IDNO: 39734; R27 3 6 Tetravalent vaccine; protamine formulated 240 μg d 0,d 21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.d.GII.4-031693-USA-2003 2 × 200 μl SEQ ID NO: 39716; R4 + GII.4-2006b092895-USA-2008 SEQ ID NO: 39729: R26 + GII.4-GZ2010-L87-Guangzhou-2011SEQ ID NO: 39734; R27 4 6 Monovalent vaccine: LNP formulated 60 μg d 0,d 21 GII.4-2006b 092895-USA-2008 i.m. SEQ ID NO: 39729; R26 2 × 100 μl 56 Bivalent vaccine: LNP formulated 60 μg d 0, d 21GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.m.GII.4-GZ2010-L87-Guangzhou-2011 2 × 100 μl SEQ ID NO: 39734; R27 6 6Tetravalent vaccine: LNP formulated 60 μg d 0, d 21 GII.4-USA-1997(total) SEQ ID NO: 39738; R28 + i.m. GII.4-031693-USA-2003 2 × 100 μlSEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729;R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 7 6 100%RiLa Control — d 0, d 21

6.2. Norovirus Challenge Experiment:

At day 3D days post immunization, the vaccinated and buffer-injectedcontrol pigs are challenged orally with Norovirus GII.4 (isolated fromhuman stool samples) to assess the protection against Norovirus-induceddiarrhea and fecal virus shedding. After virus challenge, rectal swapsand feces samples are collected at day 1, 3, 5, 7 and 10. Norovirusloads in rectal swaps and feces samples are determined usingquantitative PER. In addition, pigs are monitored forNorovirus-associated symptoms and fecal consistence scores are recordedto assess severity of the Norovirus infection.

Example 7: Immunization of Non-Human Primates and Evaluation of ImmuneResponses

Non-human primates (NHPs) are immunized with protamine or LNP formulatedmRNA vaccines with doses, application routes and vaccination schedulesas indicated in Table 8. Analysis of immune responses is performedessentially as described in Example 4 (ELISA, HGBA, and ICS).

TABLE 8 Vaccination regimen of NHPs (Example 7) Group Number of NHPsTreatment Dose/Route Vaccination schedule 1 6 Monovalent vaccineprotamine formulated 240 μg d 0, d 21 GII.4-2006b 092895-USA-2008 i.d.SEQ ID NO: 39729; R26 2 × 200 μl 2 6 Bivalent vaccine: protamineformulated 240 μg d 0, d 21 GI.1-USA-1968-Capsidprotein (total) SEQ IDNO: 39725; R29 + i.d. GII.4-GZ2010-L87-Guangzhou-2011 2 × 200 μl SEQ IDNO: 39734; R27 3 6 Tetravalent vaccine: protamine formulated 240 μg d 0,d 21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.d.GII.4-031693-USA-2003 2 × 200 μl SEQ ID NO: 39716; R4 + GII.4-2006b092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011SEQ ID NO: 39734; R27 4 6 Monovalent vaccine: LNP formulated 60 μg d 0,d 21 GII.4-2006b 092895-USA-2008 i.m. SEQ ID NO: 39729: R26 2 × 100 μl 56 Bivalent vaccine: LNP formulated 60 μg d 0, d 21GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.m.GII.4-GZ2010-L87-Guangzhou-2011 2 × 100 μl SEQ ID NO: 39734; R27 6 6Tetravalent vaccine: LNP formulated 60 μg d 0, d 21 GII.4-USA-1997(total) SEQ ID NO: 39738; R28 + i.m. GII.4-031693-USA-2003 2 × 100 μlSEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729;R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 7 6 100%RiLa Control i.m. d 0, d 21 1 × 100 μl

Example 8: Development of a Multivalent Norovirus mRNA Vaccine

8.1. Generation of Bivalent, Tetravalent and Multivalent Norovirus mRNAVaccines

For bivalent and tetravalent Norovirus mRNA vaccines, each mRNAconstruct is individually produced (as described in Example 1).

Multivalent Norovirus vaccine compositions are produced according toprocedures as disclosed in the PCT application PCT/EP2016/082487. Inshort, Norovirus DNA constructs (each of which comprising differentnorovirus coding sequences and a T7 promotor; e.g. synthetic DNAtemplates immobilized on a chip) are used as a matrix for simultaneousPER amplification. The obtained PER product mixture is purified and usedas a template for simultaneous RNA in vitro transcription to generate amixture of Norovirus mRNA constructs. The obtained Norovirus mRNAmixture is subjected to quantitative and qualitative measurements (e.g.,RNA AGE, RT-qPCR, NGS, and Spectrometry). Following that, purificationand formulation is performed (protamine formulation and LNPformulation). For the preparation of multivalent mRNA mixtures,Norovirus sequences as provided in Table 3 (see specifications) areused.

The produced bivalent, tetravalent and multivalent Norovirus mRNAvaccines are used for in vitro and in vivo experiments.

8.2. Expression Analysis of Multivalent Norovirus mRNA Vaccines UsingQuantitative Mass Spectrometry

Hela cells are transfected with bivalent, tetravalent and multivalentmRNA mixtures (see Table 9) and protein expression is analyzed usingquantitative mass spectrometry to show that every mRNA comprised in therespective mRNA mixture is efficiently translated into Norovirusprotein/antigen.

8.3. Immunization of Mice and Evaluation of Norovirus Specific ImmuneResponses

Female BALB/c mice are with protamine or LNP formulated monovalent,bivalent, tetravalent or multivalent mRNA vaccines with doses,application routes and vaccination schedules as indicated in Table 9. Asa negative control, one group of mice is injected with buffer (ringerlactate, Rile). All animals are vaccinated on day 0, 21 and 35. Bloodsamples are collected on day 49 for the Ill determination of bindingantibody titers (using an ELISA assay), blocking antibody titers (usinga HGBA assay) and cellular immune responses (ICS) performed essentiallyas described in Example 4.

TABLE 9 Vaccination regimen of mice (Example 8) Group Number of miceTreatment Dose/Route Vaccination schedule 1 6 Monovalent vaccine:Protamine formulated 40 μg d 0, d 21, d 35 GII.4-D31693-USA-2003 i.d.SEQ ID NO: 39716; R4 2 6 Bivalent vaccine: Protamine formulated 80 μg d0, d 21, d 35 R4 or R26 or R27 or R28 + (40 μg each) R4 or R26 or R27 orR28 i.d. 3 6 Tetravalent vaccine; Protamine formulated 80 μg d 0, d 21,d 35 GII.4-USA-1997 (20 μg each) SEQ ID NO: 39738; R28 + i.d.GII.4-031693-USA-2003 SEQ ID NO: 397I6; R4 + GII.4-2006b 092895-USA-2008SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO:39734; R27 4 6 Bivalent vaccine; Protamine formulated 80 μg d 0, d 21, d35 GI.1-USA-1968-Capsidprotein (40 μg each) SEQ ID NO: 39725; R29 + i.d.R4 or R26 or R27 or R28 5 6 Tetravalent vaccine: Protamine formulated 80μg d 0, d 21, d 35 GI.1-USA-1968-Capsidprotein (20 μg each) SEQ ID NO:39725; R29 + i.d. GII.4-USA-1997 SEQ ID NO: 39738; R28 +GII.4-031693-USA-2003 SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008SEQ ID NO: 39729; R26 6 6 Multivalent: Protamine formulated. 80 μg d 0,d 21, d 35 20 constructs encoding Norovirus antigens of (total) severalgenogroups, genotypes and strains i.d. (selected from Table 3). 7 6Multivalent: Protamine formulated. 80 μg d 0, d 21, d 35 50 constructsencoding Norovirus antigens of (total) several genogroups, genotypes andstrains i.d. (selected from Table 3). 8 6 Multivalent: LNP formulated.80 μg d 0, d 21, d 35 20 constructs encoding Norovirus antigens of(total) several genogroups. genotypes and strains i.m. (selected fromTable 3). 9 6 Multivalent: LNP formulated. 80 μg d 0, d 21, d 35 50constructs encoding Norovirus antigens of (total) several genogroups,genotypes and strains i.m. (selected from Table 3). 10 6 100% RiLaControl — d 0, d 21, d 35

Example 9: Expression of Norovirus Proteins in HeLa Cells and Analysisby FACS

To determine in vitro protein expression of the constructs, HeLa cellsare transiently transfected with mRNA encoding Norovirus antigens andstained using suitable customized anti Norovirus-protein antibodies(raised in mouse) and a FITC-coupled secondary antibody (F5262 fromSigma).

Hela cells are seeded in a 6-well plate at a density of 400000cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1%Pen/Strep), 24 h prior to transfection. Hela cells are transfected with1 and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). ThemRNA constructs are used in the experiment, including a negative controlencoding an irrelevant protein. 24 hours post transfection. Hela cellsare stained with suitable anti Norovirus-protein antibodies (raised inmouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500)and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto IIusing the FACS Diva software. Quantitative analysis of the fluorescentFITC signal is performed using the FlowJo software package (Tree Star,Inc.).

Example 10: Expression and Secretion of Norovirus Proteins Using WesternBlot

For the analysis of Norovirus protein secretion, Hela cells aretransfected with 1 μg and 2 μg unformulated mRNA (R1-R29, see Table 4)including a negative control encoding an irrelevant protein usingLipofectamine as the transfection agent. Supernatants, harvested 24hours post transfection, are filtered through a 0.2 μm filter. Clarifiedsupernatants are applied on top of 1 ml 20% sucrose cushion (in PBS) andcentrifuged at 14000 rcf (relative centrifugal force) for 2 hours at 4°C. Horn virus protein content is analyzed by Western Blot suitablecustomized anti Norovirus-protein antibodies (raised in mouse; 1:500diluted) as primary antibody in combination with secondary anti mouseantibody coupled to IRDye 800CW (Licor Biosciences). The presence ofαβ-tubulin is also analyzed as control for cellular contamination(αβ-tubulin: Cell Signalling Technology; 1:1000 diluted) in combinationwith secondary anti rabbit antibody coupled to IRDye 680RD (LicorBiosciences).

For the analysis of Norovirus proteins in cell lysates, Hale cells aretransfected with 1 μg and 2 μg unformulated mRNAs (R1-R29, see Table 4)including a negative control encoding an irrelevant protein usingLipofectamine as the transfection agent 24 hours post transfection, Heldcells are detached by trypsin-free/EDTA buffer, harvested, and celllysates are prepared. Cell lysates are subjected to SOS-PAGE undernon-denaturating/non-reducting followed by western blot detection.Western Blot analysis is performed using a suitable customized antiNorovirus-protein antibodies antibody (raised in mouse; 1:500 diluted)as primary antibody in combination with secondary anti mouse antibodycoupled to IRDye 800CW (Licor Biosciences).

Example 11: Preparation of Norovirus Vaccine Compositions

For in vivo vaccination experiments, different compositions of NorovirusmRNA vaccine are prepared using Norovirus mRNA constructs (see Table 4).One composition comprises protamine-complexed mRNA, one compositioncomprises mRNA that is formulated with an aluminum phosphate adjuvant.

11.1. Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”;RNActive®):

Norovirus mRNA constructs are complexed with protamine prior to use inin vivo vaccination experiments. The mRNA complexation consists of amixture of 50% free mRNA and 50% mRNA complexed with protamine at aweight ratio of 2:1. First, mRNA is complexed with protamine by additionof protamine-Ringer's lactate solution to mRNA. After incubation for 10minutes, when the complexes are stably generated, free mRNA is added,and the final concentration of the vaccine is adjusted with Ringer'slactate solution.

11.2. Preparation of mRNA with Alum Phosphate (“Vaccine Composition 2”):

mRNA constructs are mixed with the desired amount of aluminum phosphateadjuvant in Ringer's lactate solution (“naked mRNA”).

Example 12: Vaccination of Mice and Evaluation of Norovirus SpecificImmune Response

12.1. Immunization

Female BALB/c mice are injected intradermally (i.d.) and intramuscularly(i.m.) with respective mRNA vaccine compositions (prepared according toExample 11) with doses, application routes and vaccination schedules asindicated in Table 10. As a negative control, one group of mice isvaccinated with buffer (ringer lactate). All animals are vaccinated onday 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 forthe determination of binding and neutralizing antibody titers (seebelow).

TABLE 10 Vaccination regimen (Example 12) Number Route/ VaccinationGroup of mice Vaccine composition Volume Schedule (day) 1 10 80 μgNorovirus i.d. 0/21/35 RNActive ® 2 × 50 μl Composition 1 2 10 40 μgNorovirus i.d. 0/21/35 RNActive ® 2 × 50 μl Composition 1 3 10 20 μgNorovirus i.d. 0/21/35 RNActive ® 2 × 50 μl Composition 1 4 10 40 μgNorovirus i.m. 0/21/35 naked RNA 2 × 25 μl Composition 2 5 10 40 μgNorovirus i.m. 0/21/35 naked RNA 2 × 25 μl Composition 2 6 10 40 μgNorovirus i.m. 0/21/35 naked RNA 2 × 25 μl Composition 2 7 10 100% RiLaControl i.d. 0/21/35 2 × 50 μl

12.2. Determination of Anti-Norovirus Protein Antibodies by ELISA:

ELISA is performed using inactivated Norovirus infected cell lysate forcoating. Coated plates are incubated using respective serum dilutions,and binding of specific antibodies to the Norovirus antigens aredetected using biotinylated isotype specific anti-mouse antibodiesfollowed by streptavidin-HRP (horse radish peroxidase) with ABTS assubstrate. Endpoint titers of antibodies directed against the Norovirusantigens are measured by ELISA on day 63 after three vaccinations.

12.3. Intracellular Cytokine Staining

Splenocytes from vaccinated mice are isolated according to a standardprotocol known in the art. Briefly, isolated spleens are grinded througha cell strainer and washed in PBS/1% FBS followed by red blood celllysis. After an extensive washing step with PBS/1% FBS splenocytes areseeded into 96-well plates (2×10⁶ cells per well). The cells arestimulated with a mixture of four Norovirus protein specific peptideepitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of ananti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in thepresence of a protein transport inhibitor. After stimulation, cells arewashed and stained for intracellular cytokines using theCytofix/Cytoperm reagent (BD Biosciences) according to themanufacturer's instructions. The following antibodies are used forstaining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC(1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) andincubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguishlive/dead cells (Invitrogen). Cells are acquired using a Canto II flowcytometer (Beckton Dickinson). Flow cytometry data is analyzed usingFlowJo software package (Tree Star, Inc.)

12.4. Norovirus Plaque Reduction Neutralization Test (PRNT50)

Sera are analyzed by a plaque reduction neutralization test (PRNT50),performed as commonly known in the art. Briefly, obtained serum samplesof vaccinated mice are incubated with Norovirus. That mixture is used toinfect cultured cells, and the reduction in the number of plaques isdetermined.

Example 13: Clinical Development of a Norovirus mRNA Vaccine Composition

To demonstrate safety and efficiency of the NoraviromRNA vaccinecomposition, a randomized, double blind, placebo-controlled clinicaltrial (phase I) is initiated.

For clinical development, GMP-grade RNA is produced using an establishedGMP process, implementing various quality controls on DNA level and RNAlevel as described in detail in WO 2016/180430A1.

In the clinical trial, a cohort of human volunteers is intradermally orintramuscularly injected for at least two times with a monovalent, or abivalent, or a tetravalent or a multivalent mRNA based Norovirus vaccineas specified herein.

In order to assess the safety profile of the Norovirus vaccinecompositions according to the invention, subjects are monitored afteradministration (vital signs, vaccination site tolerability assessments,hematologic analysis).

The efficacy of the immunization is analysed by determination of virusneutralizing titers (VNT) or HBGA blocking titers in sera fromvaccinated subjects. Blood samples are collected on day 0 as baselineand after completed vaccination. Sera are analyzed for virusneutralizing antibodies or HBGA blocking antibodies.

Furthermore, a subset of subjects is challenged with live GI.1 Norwalkvirus or placebo by oral administration. Subjects are followedpost-challenge for symptoms of Norovirus associated illness, infectionand immune responses. There are multiple clinical assessments andcollection of blood, emesis, saliva, and stool specimens.

Lengthy table referenced here US20190125857A1-20190502-T00001 Pleaserefer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190125857A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. Artificial nucleic acid comprising at least one coding regionencoding at least one polypeptide derived from a Norovirus, and/or afragment or variant thereof.
 2. The artificial nucleic acid according toclaim 1, wherein the at least one encoded polypeptide is selected fromthe group consisting of a non-structural protein derived from aNorovirus and/or a capsid protein derived from a Norovirus, and/or afragment or variant thereof.
 3. The artificial nucleic acid according toclaim 1 or 2, wherein the at least one encoded polypeptide is selectedfrom the group consisting of Norovirus non-structural proteins NS1/NS2,NS3, NS4, NS5, NS6, NS7, Norovirus capsid protein VP1 and Noroviruscapsid protein VP2, and/or a fragment or variant thereof.
 4. Theartificial nucleic acid according to any one of claims 1 to 3, whereinthe artificial nucleic acid is derived from a Norovirus selected fromthe group consisting of genogroup I Norovirus, genogroup II Norovirus,genogroup III Norovirus, genogroup IV Norovirus, and genogroup VNorovirus; preferably the artificial nucleic acid is derived from aNorovirus selected from the group consisting of a GI.1 to GI.17Norovirus, GII.1 to GII.24 Norovirus, GIII.1 to GIII.4 Norovirus, GIV.1to GIV.4 Norovirus and GV.1 to GV.4 Norovirus; mare preferably, theartificial nucleic acid is derived from a Norovirus selected from thegroup consisting of GI.1 Norovirus and GII.4 Norovirus, even morepreferably, the artificial nucleic acid is derived from a GII.4Norovirus, still more preferably, the artificial nucleic acid is derivedfrom a GII.4 CIN-1 Norovirus or a GII.4 Sydney Norovirus or a GII.4Sydney 2012 Norovirus.
 5. The artificial nucleic acid according to anyone of claims 1 to 4, wherein the at least one encoded polypeptidecomprises at least one Norovirus capsid protein VP1 or Norovirus capsidprotein VP2 and/or a fragment or a variant thereof.
 6. The artificialnucleic acid according to any one of claims 1 to 5, wherein the at leastone encoded polypeptide comprises at least one Norovirus capsid proteinVP1 and/or a fragment or variant thereof.
 7. The artificial nucleic acidaccording to any one of claims 1 to 6, wherein the at least one encodedpolypeptide comprises (i) at least one of the amino acid sequencesaccording to any one of SEQ ID NOs: 1-4410; and/or (ii) at least one ofthe amino acid sequences having, in increasing order of preference, atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to theamino acid sequence represented by any one of SEQ ID NOs: 1-4410; and/or(iii) an orthologue or a paralogue of any one of SEQ ID NOs: 1-39690,39713-39746; and/or a fragment or variant of any of these sequences. 8.The artificial nucleic acid according to any one of claims 1 to 7,wherein the at least one coding region comprises (i) at least one of thenucleic acid sequences according to any one of SEQ ID NOs: 4411-39590,39713-39745: and/or (ii) at least one of the nucleic acid sequenceshaving, in increasing order of preference, at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 62%, 63%, 64%, 55%, 55%, 57%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequencerepresented by any one of SEQ ID NOs: 4411-39690, 39713-39746; and/or(iii) at least one complement of the nucleic acid sequences which arecapable of hybridizing with a nucleic acid sequence comprising asequence as shown in SEQ ID NOs: 4411-39590, 39713-39746, and/or to anucleic acid encoding a polypeptide having a sequence as shown in SEQ IDNOs: 1-4410; and/or (iv) an orthologue or a paralogue of any one of SEQID NOs: 1-39690, 39713-39746; and/or a fragment or variant of any ofthese sequences.
 9. The artificial nucleic acid according to any one ofclaims 1 to 8, wherein the artificial nucleic acid is monocistronic,bicistronic or multicistronic.
 10. The artificial nucleic acid accordingto any one of claims 1 to 9, wherein the artificial nucleic acid ismonocistronic and wherein the coding region encodes a polypeptidecomprising at least two different Norovirus proteins as defined in anyone of claims 1 to 9, or a fragment or variant thereof.
 11. Theartificial nucleic acid according to any one of claims 1 to 9, whereinthe artificial nucleic acid is bi- or multicistronic and comprises atleast two coding regions, wherein the at least two coding regions encodeat least two polypeptides, wherein each of the at least two polypeptidescomprises at least one Norovirus protein as defined in any one of claims1 to 9, or a fragment or variant of any one of these proteins, whereinthe at least two polypeptides are preferably different polypeptides. 12.The artificial nucleic acid according to any one of claims 1 to 11,wherein the artificial nucleic acid is an RNA, preferably an mRNA. 13.The artificial nucleic acid according to any one of claims 1 to 12,wherein the artificial nucleic acid comprises a 5′-cap structure. 14.The artificial nucleic acid according to any one of claims 1 to 13,wherein the G/C content of the coding region of the mRNA sequence isincreased compared to the G/C content of the corresponding codingsequence of the wild type mRNA, or wherein the C content of the codingregion of the mRNA sequence is increased compared to the C content ofthe corresponding coding sequence of the wild type mRNA, or wherein thecodon usage in the coding region of the mRNA sequence is adapted to thehuman codon usage, or wherein the codon adaptation index (CAI) isincreased or maximised in the coding region of the mRNA sequence,wherein the encoded amino acid sequence of the mRNA sequence ispreferably not being modified compared to the encoded amino acidsequence of the wild type mRNA.
 15. The artificial nucleic acidaccording to any one of claims 1 to 14, wherein (i) the at least onecoding region comprises a nucleic acid sequence, which iscodon-optimized; and/or (ii) the at least one coding sequence comprisesa nucleic acid sequence, which is identical or at least 50%, 60%, 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 8821-13230, 26461-39690, 39715, 39719, 39717,39720, 39721, 39724, 39725, 39728, 39729, 39730, 39733, 39734, 39737,39738, 39741, 39742, 39745 and 39746, or a fragment or variant of any ofthese sequences; and/or (iii) the at least one coding sequence comprisesa nucleic acid sequence, which is identical or at least 50%, 60%, 70%,80%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 13231-17640, or a fragment or variant of anyof these sequences; and/or (iv) the artificial nucleic acid according toany one of the preceding claims, wherein the at least one codingsequence comprises a nucleic acid sequence, which is identical or atleast 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 17641-22050, or afragment or variant of any of these sequences; and/or (v) the artificialnucleic acid according to any one of the preceding claims, wherein theat least one coding sequence comprises a nucleic acid sequence, which isidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleicacid sequence selected from the group consisting of SEQ ID NOs:22051-26460, or a fragment or variant of any of these sequences.
 16. Theartificial nucleic acid according to any one of claims 1 to 15, whereinthe artificial nucleic acid comprises at least one histone stem-loop.17. The artificial nucleic acid according to claim 16, wherein the atleast one histone stem-loop comprises a nucleic acid sequence accordingto the following formulae (I) or (II): formula (I) (stem-loop sequencewithout stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein: stem1 or stem2 bordering elements N₁₋₆ is a consecutivesequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5,even more preferably of 3 to 5, most preferably of 4 to 5 or 5 N,wherein each N is independently from another selected from a nucleotideselected from A, U, T, G and C, or a nucleotide analogue thereof; stem1[N₀₋₂GN₃₋₅] is reverse complementary or partially reverse complementarywith element stem2, and is a consecutive sequence between of 5 to 7nucleotides; wherein N₀₋₂ is a consecutive sequence of 0 to 2,preferably of 0 to 1, more preferably of 1 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof; wherein N₃₋₅ is aconsecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of4 N, wherein each N is independently from another selected from anucleotide selected from A, U, T, G and C or a nucleotide analoguethereof, and wherein G is guanosine or an analogue thereof, and may beoptionally replaced by a cytidine or an analogue thereof, provided thatits complementary nucleotide cytidine in stem2 is replaced by guanosine;loop sequence [N₀₋₄(U/T)N₀₋₄] is located between elements stem1 andstem2, and is a consecutive sequence of 3 to 5 nucleotides, morepreferably of 4 nucleotides; wherein each No-4 is independent fromanother a consecutive sequence of 0 to 4, preferably of 1 to 3, morepreferably of 1 to 2 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof; and wherein U/T represents uridine, oroptionally thymidine; stem2 [N₃₋₅CN₀₋₂] is reverse complementary orpartially reverse complementary with element stem1, and is a consecutivesequence between of 5 to 7 nucleotides; wherein N3-5 is a consecutivesequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N,wherein each N is independently from another selected from a nucleotideselected from A, U, T, G and C or a nucleotide analogue thereof; whereinN₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1, morepreferably of 1 N, wherein each N is independently from another selectedfrom a nucleotide selected from A, U, T, G and C or a nucleotideanalogue thereof; and wherein C is cytidine or an analogue thereof, andmay be optionally replaced by a guanosine or an analogue thereofprovided that its complementary nucleotide guanosine in stem1 isreplaced by cytidine; wherein stem1 and stem2 are capable of basepairing with each other forming a reverse complementary sequence,wherein base pairing may occur between stem1 and stem2, or forming apartially reverse complementary sequence, wherein an incomplete basepairing may occur between stem1 and stem2.
 18. The artificial nucleicacid according to claim 17, wherein the at least one histone stem-loopcomprises a nucleic acid sequence according to the following formulae(la) or (IIa): formula (Ia) (stem-loop sequence without stem borderingelements):

formula (IIa) (stem-loop sequence with stem bordering elements):


19. The artificial nucleic acid according to any one of claims 16 to 18,wherein the at least one histone stem loop comprises a nucleic acidsequence according to SEQ ID NO: 39709 to SEQ ID NO: 39710, or afragment or variant thereof.
 20. The artificial nucleic acid moleculeaccording to any one of claims 1 to 19, wherein the artificial nucleicacid comprises an untranslated region (UTR).
 21. The artificial nucleicacid according to claim 20, wherein the artificial nucleic acidcomprises a 3′-UTR.
 22. The artificial nucleic acid according to claim21, wherein the 3′-UTR comprises at least one heterologous 3′-UTRelement.
 23. The artificial nucleic acid according to claim 21 or 22,wherein the 3′-UTR comprises a poly(A) sequence and/or a poly(C)sequence.
 24. The artificial nucleic acid according to claim 23, whereinthe poly(A) sequence comprises 10 to 200, 10 to 100, 40 to 80 or 50 to70 adenosine nucleotides, and/or the poly(C) sequence comprises 10 to200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides. 25.The artificial nucleic acid according to any one of claims 1 to 24,wherein the at least one heterologous 3′-UTR element comprises a nucleicacid sequence derived from a 3′-UTR of a gene, which preferably encodesa stable mRNA, or from a homolog, a fragment or a variant of said gene.26. The artificial nucleic acid according to any one of claims 1 to 25,wherein the at least one heterologous 3′-UTR element comprises a nucleicacid sequence derived from a 3′-UTR of a gene selected from the groupconsisting of an albumin gene, an α-globin gene, a β-globin gene, atyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alphagene, or from a humping, a fragment or a variant thereof.
 27. Theartificial nucleic acid according to any one of claims 1 to 29, whereinthe at least one heterologous 3′-UTR element comprises a nucleic acidsequence derived from a 3′-UTR of an α-globin gene, preferablycomprising the corresponding RNA sequence of the nucleic acid sequenceaccording to SEQ ID NO: 39701, or SEQ ID NO: 39702, a homolog, afragment, or a variant thereof.
 28. The artificial nucleic acidaccording to any one of claims 1 to 27, wherein the at least oneheterologous 3′-UTR element comprises a nucleic acid sequence, which isderived from the 3′-UTR of a vertebrate albumin gene or from a variantthereof, preferably from the 3′-UTR of a mammalian albumin gene or froma variant thereof, more preferably from the 3′-UTR of a human albumingene or from a variant thereof, even more preferably from the 3′-UTR ofthe human albumin gene according to Genbank Accession numberNM_000477.5, or from a fragment or variant thereof.
 29. The artificialnucleic acid according to any one of claims 1 to 28, wherein the atleast one heterologous 3′-UTR element comprises a nucleic acid sequenceaccording to any one of SEQ ID NO: 39703 to SEQ ID NO: 39708, or ahomolog, a fragment or a variant thereof.
 30. The artificial nucleicacid according to any one of claims 1 to 29, wherein the artificialnucleic acid comprises a 5′-UTR.
 31. The artificial nucleic acidsequence according to any one of claims 1 to 30, wherein the 5′-UTRcomprises at least one heterologous 5′-UTR element.
 32. The artificialnucleic acid according to any one of claims 1 to 31, wherein the atleast one heterologous 5′-UTR element comprises a nucleic acid sequence,which is derived from the 5′-UTR of a TOP gene, preferably from acorresponding RNA sequence, or a homolog, a fragment, or a variantthereof, preferably lacking the 5′TOP motif.
 33. The artificial nucleicacid according to any one of claims 1 to 32, wherein the at least oneheterologous 5′-UTR element comprises a nucleic acid sequence, which isderived from a 5′-UTR of a TOP gene encoding a ribosomal protein,preferably from a corresponding RNA sequence, or from a homolog, afragment or a variant thereof, preferably lacking the 5′TOP motif. 34.The artificial nucleic acid according to any one of claims 1 to 33,wherein the at least one heterologous 5′-UTR element comprises a nucleicacid sequence, which is derived from a 5′-UTR of a TOP gene encoding aribosomal Large protein (RPL), preferably RPL32 or RPL35A, or from agene selected from the group consisting of HSD17B4, ATP5A1, AIG1, ASAHI,COX6C or ABCB7 (MDR), or from a homolog, a fragment or variant of anyone of these genes, preferably lacking the 5′TOP motif.
 35. Theartificial nucleic acid according to any one of claims 1 to 34, whereinthe at least one heterologous 5′-UTR element comprises a nucleic acidsequence according to SEQ ID NO: 39691 to SEQ ID NO: 39694, or ahomolog, a fragment or a variant thereof.
 36. The artificial nucleicacid according to any one of claims 1 to 35 comprising, preferably in 5′to 3′ direction, the following elements: a) optionally a 5′-capstructure, preferably m7GpppN, b) a coding region encoding at least onepolypeptide derived from a Norovirus as described herein, preferablyVP1, or a fragment or variant thereof, c) optionally a poly(A) tail,preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70adenosine nucleotides, d) optionally a poly(C) tail, preferablyconsisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40cytosine nucleotides, and e) optionally a histone stem-loop, preferablycomprising the RNA sequence according to SEQ ID NO: 39709 to SEQ ID NO:39710.
 37. The artificial nucleic acid according to any one of claims 1to 36 comprising, preferably in 5′ to 3′ direction, the followingelements: a) optionally a 5′-cap structure, preferably m7GpppN, b) acoding region encoding at least one polypeptide derived from aNorovirus, preferably VP1 as described herein, or a fragment or variantthereof, c) a 3′-UTR element comprising a nucleic acid sequence, whichis derived from an α-globin gene, preferably comprising thecorresponding RNA sequence of the nucleic acid sequence according to SEQID NO: 39701, or SEQ ID NO: 39702, or a homolog, a fragment or a variantthereof, d) optionally a poly(A) tail, preferably consisting of 10 to200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, e)optionally a poly(C) tail, preferably consisting of 10 to 200, 10 to100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and f)optionally a histone stem-loop, preferably comprising the RNA sequenceaccording to SEQ ID NO: 39709 to SEQ ID NO:
 39710. 38. The artificialnucleic acid according to any one of claims 1 to 37, wherein theartificial nucleic acid comprises a nucleic acid sequence according toany one of SEQ ID NOs: 39713-39746, preferably a nucleic acid sequenceaccording to any one of SEQ ID NOs: 39716, 39721, 39729, 39734, 39738,39725, or a fragment or variant of any of these sequences.
 39. Theartificial nucleic acid according to any one of claims 1 to 38,comprising, preferably in 5′ to 3′ direction, the following elements: a)optionally a 5′-cap structure, preferably m7GpppN, b) a 5′-UTR element,which comprises or consists of a nucleic acid sequence, which is derivedfrom the 5′-UTR of a TOP gene, preferably comprising a nucleic acidsequence according to SEQ ID NO: 39691, or SEQ ID NO: 39692, or ahoming, a fragment or a variant thereof, c) a coding region encoding atleast one polypeptide derived from a Norovirus, preferably VP1 asdescribed herein, or a fragment or variant thereof, d) a 3′-UTR elementcomprising a nucleic acid sequence, which is derived from an albumingene, preferably comprising the corresponding RNA sequence of thenucleic acid sequence according to SEQ ID NO: 39705, or SEQ ID NO:39706, or a humping, a fragment or a variant thereof, e) optionally apoly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or50 to 70 adenosine nucleotides, f) optionally a poly(C) tail, preferablyconsisting of 10 to 200, 10 to 100, 20 to 70, 20 to 90 or 10 to 40cytosine nucleotides, and g) optionally a histone stem-loop, preferablycomprising the RNA sequence according to SEQ ID NO: 39709 to SEQ ID NO:39710.
 40. The artificial nucleic acid according to any one of claims 1to 39, wherein the artificial nucleic acid comprises a nucleic acidsequence according to any one of SEQ ID NOs: 39713-39749, preferably anucleic acid sequence according to any one of SEQ ID NOs: 39716, 39721,39729, 39734, 39738, 39725, or a fragment or variant of any of thesesequences.
 41. The artificial nucleic acid according to any one ofclaims 1 to 40, wherein the coding region comprises a modified nucleicacid sequence.
 42. The artificial nucleic acid according to any one ofclaims 1 to 41, wherein the at least one coding region comprises anucleic acid sequence encoding a molecular tag and wherein the moleculartag is selected from the group consisting of a FLAG tag, aglutathione-S-transferase (GST) tag, a His tag, a Myc tag, an E tag, aStrep tag, a green fluorescent protein (GFP) tag and an HA tag. 43.Composition comprising at least one artificial nucleic acid as definedby any one of claims 1 to 42 and a pharmaceutically acceptable carrier.44. The composition according to claim 43, wherein the at least one mRNAis complexed with one or more cationic or polycationic compounds,preferably with cationic or polycationic polymers, cationic orpolycationic peptides or proteins, e.g. protamine, cationic orpolycationic polysaccharides and/or cationic or polycationic lipids. 45.The composition according to any one of claims 43 to 44, wherein the N/Pratio of the at least one mRNA to the one or more cationic orpolycationic compounds is in the range of about 0.1 to 20, including arange of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and ofabout 0.7 to 1.5.
 46. The composition according to any one of claims 43to 45 comprising the at least one mRNA, which is complexed with one ormore cationic or polycationic compounds, and at least one free mRNA. 47.The composition according to any one of claims 43 to 46, wherein the atleast one complexed mRNA is identical to the at least one free mRNA. 48.The composition according to any one of claims 43 to 47, wherein themRNA is complexed with one or more lipids, thereby forming liposomes,lipid nanoparticles and/or lipoplexes.
 49. The composition according toany one of claims 43 to 48, wherein the composition comprises at leastone adjuvant.
 50. The composition according to any one of claims 43 to49, wherein a) the composition comprises a plurality or more than one ofthe mRNA sequences each defined in any one of claims 1 to 42; or b) thecomposition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 49, 47, 48, 49,50 or more artificial nucleic acids as defined by any one of claims 1 to42, wherein each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50 or more artificial nucleic acids comprises at least one coding regionencoding at least one polypeptide comprising a Norovirus protein asdefined in any one of claims 1 to 42, and/or a fragment or a variant ofany one of these proteins, wherein each coding region preferably encodesa different Norovirus protein, more preferably each coding regionencodes a capsid protein, preferably VP1 of a different Norovirus. 51.The composition according to any one of claims 43 to 50, wherein a)wherein each of the mRNA sequences encodes at least one differentantigenic peptide or protein derived from proteins of the sameNorovirus; and/or b) the composition comprises at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more artificial nucleic acids as definedby any one of claims 1 to 42, wherein each of the at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50 or more artificial nucleic acidscomprises at least one coding region encoding at least one polypeptidecomprising at least two different Norovirus proteins, preferably VP1 andVP2, as defined in any one of claims 1 to 42, and/or a fragment or avariant of any one of these proteins.
 52. The composition according toany one of claims 43 to 51, wherein the at least one artificial nucleicacid is complexed at least partially with a cationic or polycationiccompound and/or a polymeric carrier, preferably a cationic protein orpeptide.
 53. The composition according to any one of claims 43 to 52,wherein (i) the ratio of complexed nucleic acid to free nucleic acid isselected from a range of about 5:1 (w/w) to about 1:10 (w/w), morepreferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even morepreferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3(w/w), wherein the ratio is most preferably about 1:1 (w/w); or (ii) themRNA is complexed with one or more cationic or polycationic compounds ina weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w) of mRNA to cationic or polycationic compound and/orwith a polymeric carrier; or optionally in a nitrogen/phosphate ratio ofmRNA to cationic or polycationic compound and/or polymeric carrier inthe range of about 0.1-10, preferably in a range of about 0.3-4 or0.3-1, and most preferably in a range of about 0.5-1 or 0.7-1, and evenmost preferably in a range of about 0.3-0.9 or 0.5-0.9; and/or whereinthe at least one artificial nucleic acid or mRNA is complexed with oneor more cationic or polycationic compounds, preferably with cationic orpolycationic polymers, cationic or polycationic peptides or proteins,e.g. protamine, cationic or polycationic polysaccharides and/or cationicor polycationic lipids and/or wherein the at least one artificialnucleic acid or mRNA is complexed with one or more lipids and therebyforming liposomes, lipid nanoparticles and/or lipoplexes.
 54. Thecomposition according to any one of claims 43 to 53 wherein thecomposition comprises (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreartificial nucleic acids as defined in claims 1 to 42; or (ii) at least10, 15, 20 or 50 artificial nucleic acids as defined in claims 1 to 42;or (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificial nucleicacids as defined in claims 1 to 42; and a pharmaceutically acceptablecarrier, wherein preferably the artificial nucleic acid encodes a capsidprotein VP1 derived from a Norovirus.
 55. The composition according toany one of claims 43 to 54, wherein (i) the artificial nucleic acids arederived from a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45,47, 48, 49, 50 or more different GI Noroviruses; or (ii) the artificialnucleic acids are derived from a single GII Norovirus or from 2, 3, 4,5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 45, 47, 48, 49, 50 or more different G11 Noroviruses; or(iv) the artificial nucleic acids are derived from a single GIIINorovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 19,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or moredifferent GIII Noroviruses; or (iv) the artificial nucleic acids arederived from a single GIV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29.27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 or more different GIV Noroviruses; or (v) the artificialnucleic acids are derived from a single BV Norovirus or from 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 19, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32.33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 49, 47, 48, 49, 50 or more different GV Noroviruses; or (vi)the artificial nucleic acids are derived from a single GI Norovirus andadditionally from a single GII Norovirus, GIII Norovirus, GIV Norovirusand/or GV Norovirus; or (vii) the artificial nucleic acids are derivedfrom a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50 or more different GI Noroviruses and additionally from a singleGII, GIII, GIV or GV Norovirus and/or from 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 and/or more GII, GIII, GIV or GU Noroviruses; whereinpreferably the artificial nucleic acids encode a capsid protein VP1derived from a Norovirus.
 56. The composition according to any one ofclaims 43 to 55, wherein (i) the artificial nucleic acids are derivedfrom a single GI.1 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50 or more different GI.1 Noroviruses; or (ii) the artificialnucleic acids are derived from a single GII.4 Norovirus or from 2, 3, 4,5, 6, 7.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GII.4 Noroviruses;or (iii) the artificial nucleic acids are derived from a single GI.1Norovirus and additionally from a single GII.4 Norovirus; or (iv) theartificial nucleic acids are derived from a single GI.1 Norovirus orfrom 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22.23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI.1Noroviruses and additionally from a single GII.4 Norovirus or from 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more GII.4 Noroviruses; and/orwherein (i) at least one of the nucleic acid sequences according to anyone of SEQ ID NO: 39713 to SEQ ID NO: 39746; and/or (ii) at least one ofthe nucleic acid sequences having, in increasing order of preference, atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 96%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to thenucleic acid sequence represented by any one of SEQ ID NO: 39713 to SEQID NO: 39746; and/or (iii) at least one complement of the nucleic acidsequences which are capable of hybridizing with a nucleic acid sequencecomprising a sequence as shown in SEED NO: 39713 to SEQ ID NO: 39746;and/or (iv) an orthologue or a paralogue of any one of SEQ ID NO: 39713to SEQ ID NO: 39748; and/or a fragment or variant of any of thesesequences.
 57. Polypeptide encoded by the artificial nucleic acidaccording to any one of claims 1 to
 42. 58. Polypeptide according to anyone of claims 1 to 42 comprising at least one protein selected from thegroup consisting of NS1/NS2, NS3, NS4, NS5, NS6, NS7, VP1, and VP2derived from Norovirus, or a fragment or variant of any of theseproteins, and at least one amino acid sequence selected from the groupconsisting of: a) an amino acid sequence derived from a C-terminalfragment from mature Norovirus capsid protein VP1, or a variant thereof,wherein the C-terminal fragment consists of 3 to 20 amino acid residues,b) an amino acid sequence derived from a signal sequence of Noroviruscapsid protein VP1, or a fragment or variant thereof, and c) an aminoacid sequence derived from an N-terminal fragment from mature Norovirusnon-structural protein NS1/NS2, NS3, NS4, NS5, NS6, or NS7, or a variantthereof, wherein the N-terminal fragment consists of 3 to 20 amino acidresidues.
 59. The polypeptide according to any one of claims 57 to 58comprising a molecular tag, wherein the molecular tag is selected fromthe group consisting of a FLAG tag, a glutathione-S-transferase (GST)tag, a His tag, a Myc tag, an E tag, a Strep tag, a green fluorescentprotein (GFP) tag and an HA tag.
 60. Composition comprising thepolypeptide according to any one of claims 57 to 59, and apharmaceutically acceptable carrier.
 61. Vaccine comprising theartificial nucleic acid according to any one of claims 1 to 42, thecomposition according to any one of claims 43 to 56, the polypeptideaccording to any one of claims 57 to 59, and/or the compositionaccording to claim
 60. 62. The vaccine according to claim 61, whereinthe artificial nucleic acid according to any one of claims 1 to 42, thecomposition according to any one of claims 43 to 56, the polypeptideaccording to any one of claims 57 to 59, or the composition according toclaim 60 elicits an adaptive immune response.
 63. The vaccine accordingto claims 61 to 62, wherein the vaccine further comprises apharmaceutically acceptable carrier.
 64. The vaccine according to anyone of claims 61 to 63 further comprising an adjuvant.
 65. The vaccineaccording to any one of claims 61 to 64, wherein the vaccine ismultivalent and comprises (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore artificial nucleic acids as defined in claims 1 to 42; or (ii) atleast 10, 15, 20 or 50 artificial nucleic acids as defined in claims 1to 42; or (iii) 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 artificialnucleic acids as defined in claims 1 to
 43. 66. The vaccine according toany one of claims 61 to 65, wherein (i) the artificial nucleic acids arederived from a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 or more different GI Noroviruses; or (ii) the artificialnucleic acids are derived from a single GII Norovirus or from 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GII Noroviruses; or(iii) the artificial nucleic acids are derived from a single GIIINorovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or moredifferent GIII Noroviruses; or (iv) the artificial nucleic acids arederived from a single GIV Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 or more different GIV Noroviruses; or (v) the artificialnucleic acids are derived from a single GV Norovirus or from 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50 or more different GV Noroviruses; or (vi)the artificial nucleic acids are derived from a single GI Norovirus andadditionally from a single GII Norovirus, GIII Norovirus, GIV Norovirusand/or DV Norovirus; or (vii) the artificial nucleic acids are derivedfrom a single GI Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30.31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50 or more different GI Noroviruses and additionally from asingle GII, GIII, GIV and/or GV Norovirus or from 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50 or more GII, GIII, GIV and/or GV Noroviruses. 67.The vaccine according to any one of claims 61 to 66, wherein (i) theartificial nucleic acids are derived from a single GI.1 Norovirus orfrom 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different GI.1Noroviruses; or (ii) the artificial nucleic acids are derived from asingle GII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50 or more different GII.4 Noroviruses; or (iii) the artificial nucleicacids are derived from a single GI.1 Norovirus and additionally from asingle GII.4 Norovirus; or (iv) the artificial nucleic acids are derivedfrom a single GI.1 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50 or more different GI.1 Noroviruses and additionally from a singleGII.4 Norovirus or from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 ormore GII.4 Noroviruses.
 68. Kit or kit of parts comprising theartificial nucleic acid according to any one of claims 1 to 42, thecomposition according to any one of claims 43 to 56, the polypeptideaccording to any one of claims 57 to 59, the composition according toclaim 60 or the vaccine according to any one of claims 61 to 67,optionally comprising a liquid vehicle for solubilising, and optionallytechnical instructions providing information on administration anddosage of the components.
 69. The kit or kit of parts according to claim68 comprising Ringer lactate solution.
 70. The artificial nucleic acidaccording to any one of claims 1 to 42, the composition according to anyone of claims 43 to 56, the polypeptide according to any one of claims57 to 59, the composition according to claim 60, the vaccine accordingto any one of claims 61 to 67, or the kit or kit of parts according toclaims 68 to 69 for use as a medicament.
 71. The artificial nucleic acidaccording to any one of claims 1 to 42, the composition according to anyone of claims 43 to 56, the polypeptide according to any one of claims57 to 59, the composition according to claim 60, the vaccine accordingto any one of claims 61 to 67, or the kit or kit of parts according toclaims 68 to 69 for use in the treatment or prophylaxis of an infectionwith Norovirus or a disorder related to an infection with Norovirus. 72.The artificial nucleic acid according to any one of according to any oneof claims 1 to 42, the composition according to any one of claims 43 to56, the polypeptide according to any one of claims 57 to 59, thecomposition according to claim 60, the vaccine according to any one ofclaims 61 to 67, or the kit or kit of parts according to claims 68 to69, wherein the artificial nucleic acid, the composition, the vaccine orthe active component of the kit or kit of parts is administered byinjection, preferably by needle-less injection, more preferably by jetinjection.
 73. The artificial nucleic acid according to any one ofclaims 1 to 42, the composition according to any one of claims 43 to 56,the polypeptide according to any one of claims 57 to 59, the compositionaccording to claim 60, the vaccine according to any one of claims 61 to67, or the kit or kit of parts according to claim 68 to 69 for useaccording to any one of claims 70 to 72, wherein the treatment orprophylaxis comprises the administration of a further activepharmaceutical ingredient.
 74. Method of treating or preventing adisorder, wherein the method comprises administering to a subject inneed thereof the artificial nucleic acid according to any one of claims1 to 42, the composition according to any one of claims 43 to 56, thepolypeptide according to any one of claims 57 to 59, the compositionaccording to claim 60, the vaccine according to any one of claims 61 to67, or the kit or kit of parts according to claims 68 to
 69. 75. Themethod according to claim 74, wherein the disorder is an infection withNorovirus or a disorder related to an infection with Norovirus.